DE112013003420T5 - Modified Clostridium strain for butanol production - Google Patents

Abstract

The present invention relates to improved thiolase variants as well as microorganisms expressing them and methods using them. Such processes can be used for improved production of butanol. Preferably, the improved thiolase variants have thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of wild-type thiolA from C. acetobutylicum.

Description

FIELD OF THE INVENTION

The present invention relates to the field of industrial microbiology and genetic modification of microorganisms. More particularly, the invention relates to a process for preparing a chemical compound using an acetoacetyl-coenzyme A-dependent pathway in a microbial host, such as the genus Clostridium, wherein the chemical is in particular n-butanol.

BACKGROUND OF THE INVENTION

1-butanol (n-butanol or simply butanol) is an important utility chemical that today is made almost exclusively from petrochemical raw materials. The fermentative production of butanol is limited to present knowledge on the genus Clostridium, a group of Gram-positive, spore-forming anaerobes, which mainly includes species from terrestrial habitats (drought in 1998, drought in 2011). The biosynthesis of butanol requires a C ₄ -specific biosynthetic pathway, which deviates from a central metabolic intermediate, such as the activated C ₂ compound acetyl-CoA. Such formation of carbon-carbon bonds is an important metabolic step and the thiolase is a common biocatalyst that mediates this reaction.

Clostridium acetobutylicum was the first to be discovered among solventogenic clostridines and is still the best characterized solventogenic Clostridium species. Improvements of Clostridium strains for biotechnological applications have been obtained using random mutagenesis and plate screening approaches ( US 4,757,010 ; US 6,358,717 ). In light of the recent development of new tools for metabolism and analysis for members of the genus Clostridium, several rational approaches to improving butanol production have been made (Lee et al., 2008, Papoutsakis, 2008, Lütke-Eversloh & Bahl, 2011). However, these are limited to the manipulation of gene expression patterns, such as gene knockout or down-regulation and / or gene overexpression, for the manipulation of metabolic currents towards the desired product.

Thiolase (acetyl-coenzyme A [CoA] acetyltransferase, EC 2.3.1.19) plays a key role in the production of butanol and other metabolites in organisms, including C. acetobutylicum, and its activity may increase the ratio of C3- & C4 products ( eg, butanol, acetone and butyrate) to C2 products (eg, ethanol and acetate); (Wiesenborn et al., (1988)). Wiesenborn et al. (1988) further described that thiolase can be inhibited by micromolar levels of coenzyme A [CoA].

In one of Stiller et al. (2008), which aimed to direct the flow of acetate toward butanol formation, the aldehyde / alcohol dehydrogenase gene adhE1 (adh) and the thiolase gene thlA (thl) in the solvent-negative degenerate strain C. acetobutylicum M5 homologously overexpressed. While adhE1 overexpression resulted in increased titers of the sum of all alcohols (including ethanol and butanol), additional thlA expression could not alter the phenotype that acetate is the major fermentation product in strain C. acetobutylicum M5. In addition, butanol production of a C. acetobutylicum strain ATCC 824, which comprises a downregulated acetone pathway and adhE1 overexpression, could not be increased by concomitant thlA overexpression (Sillers et al., 2008, Sillers et al., 2009). , These findings clearly indicated that increased levels of thlA transcripts do not directly lead to increased current in the C ₄ pathway.

In conclusion, it is desirable to obtain bacteria capable of producing more C3 and C4 products (such as butanol), but that altering gene expression is obviously not sufficient to meet this desire.

PROBLEM TO BE SOLVED

It is an object of the present invention to provide an improved process for the microbial production of butanol and an improved enzyme suitable for use in the process. It is especially desired that the improved enzyme be useful as a biocatalyst for microbial butanol production and, as compared to prior art enzymes, especially under physiological conditions, i.e.. H. when expressed in a strain capable of producing butanol and / or other C3 / C4 products, exhibits improved performance. Other objects of the invention which have been achieved will be apparent from the following description of the invention.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides an improved process for producing a chemical using an acetoacetyl-CoA dependent pathway. This method is improved because it has a thiolase activity with higher specific activity and / or with reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of a wild-type enzyme such as the polypeptide of SEQ ID NO: 1. In a preferred embodiment, the thiolase variants of the invention exhibit reduced sensitivity to their physiological inhibitor coenzyme A (CoA-SH). The method of the invention can be carried out by recombinantly expressing the enzyme of the invention, resulting in a recombinant (micro) organism, such as a recombinant Clostridium acetobutylicum, which expresses the thiolase of the invention. The enzyme surprisingly shows improved butanol productivity.

The inventors arrived at the invention by analyzing the polypeptide sequence of a wild-type thiolase, preparing a library of mutated thiolase genes, followed by a high-throughput screening assay using Escherichia coli as host cells for the expression of the library of mutant thiolase genes. Screening of this library led to the identification of various thiolase enzyme variants, including a thiolase-enzyme variant with significantly increased activity in the presence of free CoA-SH. The most preferred (optimized) thiolase variant has three amino acid substitutions (R133G, H156N, G222V) and its gene was expressed in C. acetobutylicum ATCC 824 to study the effect of reduced feedback inhibition on solvent production.

In addition to significantly delayed ethanol and acetone production, the ethanol titer was increased by 46%. The butanol titer was increased by 18%, whereas the final acetone concentrations were similar to the vector control strain.

The invention also provides host cells expressing the enzyme of the invention and / or useful in the method of the invention, as well as nucleic acids encoding the enzyme of the invention.

POINTS

• [1] Ein Verfahren zur Herstellung einer Chemikalie unter Verwendung eines Acetoacetyl-CoA-abhängigen Stoffwechselweges, der eine Thiolaseaktivität mit höherer spezifischer Aktivität und/oder verringerter Sensitivität gegenüber dem Inhibitor Coenzym A im Vergleich zu der Aktivität und Sensitivität des Polypeptids mit der SEQ ID NO: 1 besitzt.
• [2] Ein Verfahren nach Punkt 1, wobei die Thiolase 5% oder höhere, vorzugsweise 10% oder höhere, stärker bevorzugt 20% oder höhere, noch stärker bevorzugt 30% oder höhere Aktivität im Vergleich zu dem parentalen Wildtyp-Enzym unter physiologischen Bedingungen besitzt.
• [3] Ein Verfahren nach einem der Punkte 1 oder 2, wobei die Aktivität der Thiolase durch 10 μM CoA-SH nicht gehemmt wird.
• [4] Ein Verfahren nach einem der Punkte 1–3, wobei die Chemikalie ein C4-Alkohol ist.
• [5] Ein Verfahren nach Punkt 4, wobei die Chemikalie Butanol, vorzugsweise n-Butanol, ist.
• [6] Ein Verfahren nach einem der Punkte 1–5, wobei die Produktion in einer Wirtszelle erfolgt, die zur Gattung Clostridium gehört.
• [7] Ein Verfahren nach Punkt 6, wobei eine solche Wirtszelle zu einer der folgenden Spezies gehört: C. acetobutylicum, C, beijerinckii, C. saccharoperbutylacetonicum, C. butyricum, C. pasteurianum, C. saccharobutylicum, C. Ijungdahlii, C. thermocellum, C. thermobutyricum, C. cellulolyticum.
• [8] Ein Verfahren nach einem der Punkte 1–7, wobei Butanol durch nicht-natürliche Butanol-produzierende Stämme hergestellt wird.
• [9] Ein Verfahren nach einem der Punkte 1–8, wobei Butanol durch einen Stamm produziert wird, der zur Gattung Escherichia, Bacillus, Lactobacillus, Pseudomonas, Ralstonia, Saccharomyces gehört.
• [10] Ein Verfahren nach einem der Punkte 1–9, wobei Butanol durch einen Stamm produziert wird, der zur Spezies E. coli, B. subtilis, L. brevis, L. plantarum, P. putida, P. fluorescens, P. aeruginosa, R. eutropha, S. cerevisiae gehört.
• [11] Ein Polypeptid mit Thiolaseaktivität, dadurch gekennzeichnet, dass es 70% oder mehr, vorzugsweise 75% oder mehr, stärker bevorzugt 80% oder mehr, stärker bevorzugt 85% oder mehr, stärker bevorzugt 90% oder mehr, stärker bevorzugt 95% oder mehr, wie 96% oder mehr, 97% oder mehr, 98% oder mehr, 99% oder mehr Sequenzidentität mit der SEQ ID NO: 1 hat, unter der Voraussetzung, dass mindestens eine, wie eine beliebige Kombination von zwei oder alle drei der Reste 133, 156, 222 eine Mutation (ausgewählt aus Substitution, Deletion oder Insertion) in Bezug auf die SEQ ID NO: 1 aufweisen.
• [12] Das Polypeptid nach Punkt 11, wobei die Mutation(en) ausgewählt aus
• • einer beliebigen,
• • einer beliebigen Kombination von zwei und
• • vorzugsweise allen drei (SEQ ID NO: 2) der folgenden Substitutionen: R133G, H156N, G222V.
• [13] Das Verfahren nach einem der Punkte 1 bis 10, dadurch gekennzeichnet, dass die Thiolaseaktivität durch das Enzym nach einem der Ansprüche 11 bis 12 bereitgestellt wird.
• [14] Eine Nukleinsäure, die ein Polypeptid codiert, das aus der Gruppe ausgewählt ist, bestehend aus (a) dem Polypeptid nach einem der Punkte 11 oder 12 und (b) dem in einem der Ansprüche 1 bis 10 definierten Enzym mit der Thiolaseaktivität.
• [15] Ein Vektor, umfassend die Nukleinsäure nach Punkt 14.
• [16] Eine Wirtszelle, die die Nukleinsäure nach Punkt 14 oder den Vektor nach Anspruch 15 birgt.

The following points are encompassed by the present invention

• [1] A method of producing a chemical using an acetoacetyl-CoA dependent pathway which has a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of the polypeptide of SEQ ID NO : 1 owns.
• [2] A method according to item 1, wherein the thiolase has 5% or higher, preferably 10% or higher, more preferably 20% or higher, even more preferably 30% or higher activity compared to the parental wild-type enzyme under physiological conditions ,
• [3] A method according to any one of items 1 or 2, wherein the activity of the thiolase is not inhibited by 10 μM CoA-SH.
• [4] A method according to any one of items 1-3, wherein the chemical is a C4 alcohol.
• [5] A method according to item 4, wherein the chemical is butanol, preferably n-butanol.
• [6] A method according to any one of items 1-5, wherein the production takes place in a host cell belonging to the genus Clostridium.
• [7] A method according to item 6, wherein such a host cell belongs to one of the following species: C. acetobutylicum, C, beijerinckii, C. saccharoperbutylacetonicum, C. butyricum, C. pasteurianum, C. saccharobutylicum, C. Ijungdahlii, C. thermocellum, C. thermobutyricum, C. cellulolyticum.
• [8] A method according to any one of items 1-7, wherein butanol is produced by non-natural butanol producing strains.
• [9] A method according to any one of items 1-8, wherein butanol is produced by a strain belonging to the genus Escherichia, Bacillus, Lactobacillus, Pseudomonas, Ralstonia, Saccharomyces.
• [10] A method according to any one of items 1-9, wherein butanol is produced by a strain belonging to the species E. coli, B. subtilis, L. brevis, L. plantarum, P. putida, P. fluorescens, P. aeruginosa, R. eutropha, S. cerevisiae.
• [11] A polypeptide having thiolase activity, characterized in that it is 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, such as 96% or more, 97% or more, 98% or more, 99% or more sequence identity to SEQ ID NO: 1, provided that at least one, like any combination of two or all three of the residues 133, 156, 222 a mutation (selected from substitution, deletion or insertion) with respect to SEQ ID NO: 1.
• [12] The polypeptide of item 11, wherein the mutation (s) is selected from
• • any,
• • any combination of two and
• Preferably all three (SEQ ID NO: 2) of the following substitutions: R133G, H156N, G222V.
• [13] The method according to any one of items 1 to 10, characterized in that the thiolase activity is provided by the enzyme according to any one of claims 11 to 12.
• [14] A nucleic acid encoding a polypeptide selected from the group consisting of (a) the polypeptide of any one of items 11 or 12 and (b) the enzyme having the thiolase activity as defined in any one of claims 1 to 10.
• [15] A vector comprising the nucleic acid of item 14.
• [16] A host cell harboring the nucleic acid of item 14 or the vector of claim 15.

• [1] Ein Verfahren zur Herstellung einer Chemikalie unter Verwendung eines Acetoacetyl-CoA-abhängigen Stoffwechselweges, der eine Thiolaseaktivität mit höherer spezifischer Aktivität und verringerter Sensitivität gegenüber dem Inhibitor Coenzym A im Vergleich zu der Aktivität und Sensitivität des Polypeptids mit der SEQ ID NO: 1 besitzt und wobei die Aktivität dieser Thiolase durch 10 μM CoA-SH nicht gehemmt wird.
• [2] Ein Verfahren zur Herstellung einer Chemikalie unter Verwendung eines Acetoacetyl-CoA-abhängigen Stoffwechselweges, der eine Thiolaseaktivität mit höherer spezifischer Aktivität und/oder verringerter Sensitivität gegenüber dem Inhibitor Coenzym A im Vergleich zu der Aktivität und Sensitivität des Polypeptids mit der SEQ ID NO: 1 besitzt und wobei die Thiolase ein mutiertes Protein ist mit mindestens einer Mutation, ausgewählt aus
• • einer beliebigen,
• • einer beliebigen Kombination von zwei und
• • vorzugsweise allen drei (SEQ ID NO: 2) der folgenden Substitutionen: R133G, H156N, G222V.
• [3] Ein Verfahren zur Herstellung einer Chemikalie unter Verwendung eines Acetoacetyl-CoA-abhängigen Stoffwechselweges, der eine Thiolaseaktivität mit höherer spezifischer Aktivität und/oder verringerter Sensitivität gegenüber dem Inhibitor Coenzym A im Vergleich zu der Aktivität und Sensitivität des Polypeptids mit der SEQ ID NO: 1 besitzt und wobei die Thiolase ein mutiertes Protein ist, das mindestens eine Mutation in der CoA-SH-bindenden Schleifendomäne in Bezug auf das parentale Wildtyp-Enzym hat.
• [4] Ein Verfahren zur Herstellung einer Chemikalie unter Verwendung eines Acetoacetyl-CoA-abhängigen Stoffwechselweges, der eine Thiolaseaktivität mit höherer spezifischer Aktivität und/oder verringerter Sensitivität gegenüber dem Inhibitor Coenzym A im Vergleich zu der Aktivität und Sensitivität des Polypeptids mit der SEQ ID NO: 1 besitzt, wobei die Thiolase ein mutiertes Protein ist, das mindestens eine Mutation in der CoA-SH-bindenden Schleifendomäne in Bezug auf das parentale Wildtyp-Enzym hat, und wobei die Bestimmung der Thiolaseaktivität durch einen 3-Hydroxybutyryl-CoA-Dehydrogenase-gekoppelten spektralphotometrischen Assay durchgeführt wird.

In addition, the present invention provides the following items:

• [1] A method of producing a chemical using an acetoacetyl-CoA dependent pathway which has a thiolase activity with higher specific activity and reduced sensitivity to the inhibitor coenzyme A compared to the activity and sensitivity of the polypeptide of SEQ ID NO: 1 and wherein the activity of this thiolase is not inhibited by 10 μM CoA-SH.
• [2] A method for producing a chemical using an acetoacetyl-CoA dependent pathway which has a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A compared to the activity and sensitivity of the polypeptide of SEQ ID NO : 1 and wherein the thiolase is a mutant protein having at least one mutation selected from
• • any,
• • any combination of two and
• Preferably all three (SEQ ID NO: 2) of the following substitutions: R133G, H156N, G222V.
• [3] A method of producing a chemical using an acetoacetyl-CoA dependent pathway which has a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of the polypeptide of SEQ ID NO : 1 and wherein the thiolase is a mutant protein having at least one mutation in the CoA-SH binding loop domain relative to the parental wild-type enzyme.
• [4] A method of producing a chemical using an acetoacetyl-CoA dependent pathway which has a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of the polypeptide of SEQ ID NO : 1, wherein the thiolase is a mutant protein having at least one mutation in the CoA-SH binding loop domain relative to the parental wild-type enzyme, and wherein the determination of thiolase activity by a 3-hydroxybutyryl-CoA dehydrogenase Coupled spectrophotometric assay is performed.

DESCRIPTION OF THE FIGURES

• (a), Kulturen von rekombinanten E. coli-Stämmen, die Wildtyp-ThlA CAC2873 exprimieren, wurden dazu verwendet, Zellrohextrakte herzustellen, die spektralphotometrischen Messungen der Thiolaseaktivität anhand der NADPH-Abnahme bei 340 nm unterworfen wurden. Zeitverlauf der Extinktion bei 340 nm (A_{340 nm}) des gekoppelten ThlA/PhaB-Assays in Rohextrakten von E. coli BL21 (DE3) pASK-IBA3::thlA^WT.
• (b), Reproduzierbarkeit des gekoppelten ThlA/PhaB-Assays in Rohextrakten von E. coli BL21 (DE3) pASK-IBA3::thlA^WT. Verteilung von 144 unabhängigen Thiolaseaktivitätsmessungen; die gestrichelte Linie gibt den Mittelwert an. Die durchschnittliche Thiolaseaktivität von E. coli pASK-IBA3::thlA^WT betrug 71 ± 4 U/mg, was eine Standardabweichung von nur 5,2% (n = 144) darstellte. Für das Screening nach ThlA-Mutanten mit verringerter Sensitivität gegenüber freiem CoA-SH (dem physiologischen Inhibitor) wurde der Assay unter den gleichen Bedingungen mit Zugabe von jeweils 10 μM CoA-SH wiederholt, was 27 ± 1 U/mg der Wildtyp-ThlA-Aktivität ergab. Somit stellten die C. acetobutylicum-Wildtyp-Thiolaseaktivitäten in Rohextrakten von rekombinanten E. coli die Referenzwerte für das anschließende Bank-Screening dar.

1 , Development of thiolase assay in 96-well microtiter plates.

• (a) Cultures of recombinant E. coli strains expressing wild-type ThlA CAC2873 were used to prepare crude cell extracts which were subjected to spectrophotometric measurements of thiolase activity by the NADPH decrease at 340 nm. Time course of absorbance at 340 nm (A _{340 nm} ) of the coupled ThlA / PhaB assay in crude extracts of E. coli BL21 (DE3) pASK-IBA3 :: thlA ^WT .
• (b), reproducibility of the coupled ThlA / PhaB assay in crude extracts of E. coli BL21 (DE3) pASK-IBA3 :: thlA ^WT . Distribution of 144 independent thiolase activity measurements; the dashed line indicates the mean. The average thiolase activity of E. coli pASK-IBA3 :: thlA ^WT was 71 ± 4 U / mg, representing a standard deviation of only 5.2% (n = 144). For screening for ThlA mutants with reduced sensitivity to free CoA-SH (the physiological inhibitor), the assay was repeated under the same conditions with the addition of 10 μM CoA-SH each, giving 27 ± 1 U / mg of wild-type ThlA- Activity revealed. Thus, wild-type C. acetobutylicum thiolase activities in crude extracts of recombinant E. coli were the benchmark for subsequent bank screening.

2 , Amino acid sequence alignment of thiolases of C. acetobutylicum (wild-type) and Z. ramigera.

Sequence alignment was performed using Clustal ^W 2.1; "*" Stands for identical, ":" for very similar and "." For similar amino acid residues. CAC2873, thiolase of C. acetobutylicum; Z. ram., Thiolase of Z. ramigera. The catalytic histidine and cysteine residues are underlined, the residues of the loop domain involved in the CoA binding are shaded.

3 , Screening of E. coli pASK-IBA3 :: thlAMUT Bank.

Thiolase activities were measured in crude cell extracts without (a) and in the presence of 10 μM free CoA-SH (b). The relative distribution in percent of wild-type thiolase activity without (c) and in the presence of 10 μM free CoA-SH (d) is shown. A total of 1620 clones of the E. coli BL21 (DE3) pASK-IBA3 :: thlA ^MUT library were analyzed for thiolase activities and sensitivity to free CoA-SH. As illustrated in the figure, thiolase activities of mutants displayed a broad spectrum of both elevated and decreased levels compared to wild-type ThlA activity ( 3a , c), and only 56 clones (<4%) gave no thiolase activity. Interestingly, such broadly scattered values were not observed when the same clones were subjected to measurements of thiolase activity in the presence of 10 μM CoA-SH, ie 80% of the mutants showed activities within ± 20% of the wild-type ThlA activity ( 3b , d). Finally, 14 Thl ^MUT variants were identified by a significantly increased activity, ie,> 150% of Thl ^WT activity in the presence of free CoA-SH. It is noteworthy that increased levels of activity from both assays detected during screening did not correlate with each other. The arrow in (d) indicates the 14 positive clones (ThlA ^MUT variants with significantly increased activity), which were selected for further characterization.

4 , Thiolase inhibition by free CoA-SH.

Open squares and dashed line, ThlA ^WT ; filled circles and solid line, ThlA ^OPT . Mean values from six independent measurements are shown. The significantly reduced sensitivity of ThlA ^OPT to free CoA-SH compared to ThlA ^WT is shown. Remarkably, at a concentration of 50 μM CoA-SH, ThlA ^OPT showed a thiolase activity more than 10-fold higher than ThlA ^WT .

5 , Fermentation profiles of recombinant C. acetobutylicum strains.

Closed circles, C. acetobutylicum pT :: thlA ^OPT ; open diamonds, C. acetobutylicum pT (vector control). Averaged data from three independent repetitions for each strain are shown. C. acetobutylicum pT :: thlA ^OPT produced similar amounts of acetone but higher levels of ethanol and butanol compared to the vector control strain. The acetone and ethanol production was significantly delayed whereas the butanol synthesis started at the same time as in the control culture. Expression of thlA ^OPT resulted in significantly increased butanol production during the stationary growth phase, which increased the final butanol titer from 142 mM (10.5 g / L) to 168 mM (12.4 g / L).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved process for the production of a chemical using an acetoacetyl-CoA dependent pathway. The present invention thus relates to a process for producing a chemical in which a larger amount of current is involved in the acetoacetyl-coenzyme A-dependent metabolic pathway.

The method of the invention is improved because it has a thiolase activity with higher specific activity and / or with reduced sensitivity to the inhibitor coenzyme A as compared to the activity and sensitivity of a wild-type enzyme such as the polypeptide of SEQ ID NO: 1 , These improved variants of the enzyme thiolase thlA, which have higher activity and / or reduced feedback inhibition, are also included in the invention. Surprisingly, microbial strains expressing such an enzyme variant show higher productivity and / or selectivity in the direction of butanol production, although prior art approaches using microbial strains overexpressing such a wild type enzyme have not shown the corresponding effect. Stiller et al. (2008).

In particular, high throughput applications have been developed to identify the thiolase variants of the invention.

In the prior art it is believed that random mutagenesis of enzymes often has the disadvantage that a large proportion, if not all, of the mutants thus obtained compared to the starting variant (which may be the wild-type variant) are not improved. The reasons may include, for example, that the mutagenesis of catalytically or structurally important residues often can not be tolerated. On the other hand, detailed information about the role of each residue in the catalysis or structure of an enzyme is often unknown. Researchers therefore often feel discouraged from using random mutagenesis approaches, especially when no comprehensive structural and functional studies on the initial variant (which may be wild-type) exist.

In an attempt to circumvent the disadvantages often associated with random mutagenesis, or more specifically, to minimize the possibility of producing largely inactive mutants, and to maximize the chances of obtaining improved enzyme variants, the inventors of the present invention have a two-step procedure applied. In the first step, they aligned the amino acid sequence of CAC2873 with the Z. ramigera thiolase. This was done considering the sequence homology of Z. ramigera thiolase (for alignment see 2 ), which had previously undergone a detailed biochemical and crystallographic analysis. As a result of this alignment, the inventors expected that amino acid residues 119-249 of CAC2873 form a large loop domain that interacts with the CoA unit of the substrate (Meriläinen et al., 2009, Meriläinen et al., 2008).

In the second step, the inventors of the present invention developed a mutagenesis protocol aimed at introducing random mutations into the loop region, i. H. the region of residues 119-254, aimed, d. H. the catalytic residues Cys89, His348 and Cys378 were not involved. The correctness of the mutant library was checked by DNA sequencing of 48 randomly selected clones. The sequenced variants in the ThlA mutant library thus obtained comprised an average number of 2.5 amino acid substitutions in the region of residues 119-254.

Thus, in one aspect, the present invention relates to a two step process for the preparation of ThlA variants. Any ThlA enzyme from any organism (preferably Clostridium species) can be used as the starting sequence, which in the first step with the in 2 aligned Z. ramigera thiolase is aligned. Alignment is done with ClustalW 2.1. A preferred region into which the mutations are to be introduced is the region homologous to the region corresponding to residues 120-249 of the 2 Z. ramigera thiolase shown corresponds ("loop region"). The term "loop region", without wishing to be bound by any theory, is used simply to refer to a region homologous to the region corresponding to residues 120-249 of the art 2 shown Z. ramigera thiolase corresponds. In a preferred embodiment, CAC2873 (SEQ ID NO: 1; 2 ) the starting sequence for the mutagenesis approach, and more preferably the region selected for mutagenesis is the loop region, ie the region corresponding to residues 119-249 of CAC2873. "Region selected for mutagenesis" may mean that mutations are preferably introduced into this region or, in a preferred embodiment, mutations are introduced exclusively into that region. Thus, in a preferred embodiment, the improved thiolases of the present invention are variants of the ThlA enzyme (CAC2873) of C. acetobutylicum ATCC 824. In one embodiment, the thiolase gene thlA of C. acetobutylicum (CAC2873) is subjected to random mutagenesis. In a preferred embodiment, the CoA-SH binding loop domain is the target of amino acid replacement by random mutagenesis.

In a second step, mutations are introduced into the thiolase or its loop region. Although this is usually done by mutating the coding sequence, any desired mutant can also be obtained by direct synthesis of the desired polypeptide. The coding sequence of the enzyme to be mutagenized can often be obtained from publicly available databases. The corresponding nucleotide can then be obtained by designing PCR primers as known in the art and amplifying from a sample from the appropriate organism. Such a sample may be, for example, the entire genomic DNA or a colony of the corresponding microorganism (colony PCR). Alternatively, the coding sequence can be obtained by reverse translation of the amino acid sequence and the corresponding oligonucleotide can be synthesized in vitro. Optionally, reverse translation may be performed by taking into account the codon usage of the organism in which expression is desired. Many methods of introducing mutations are known in the art. Random methods (methods of obtaining random mutants) include, for example, error-prone PCR. Alternatively, specific mutants can be introduced by site-directed mutagenesis. A variety of random and site-directed mutagenesis approaches are described by Sambrook and Russell 2001, and any such approaches may be used. In cases where introduction of mutations into a particular region of the starting sequence (ie loop region) is desired, random mutagenesis may be by any of them For example, by introducing site-specific mutations into said particular region or by excising the region from a nucleic acid construct comprising the entire coding sequence of the parental (eg, wild-type) enzyme, using unique restriction sites, followed by random mutagenesis of the excised region and subsequent religation into a nucleic acid construct corresponding to that resulting from cleavage at the unique restriction sites. Preferably, however, random mutagenesis is performed using synthetic oligonucleotides that make up the degenerate backbone, e.g. B. as described in Example 4 and Table 1.

In this specification, mutation may mean any one or more of any agents selected from substitution, deletion, and insertion, although substitution may be preferred. In this specification, a mutant protein is any variant of a parental protein that has at least one (preferred) mutation with respect to the parental protein.

The random mutants obtained can be sequenced at this stage (for which the (plasmid) DNA encoding the variant can be isolated), although sequencing at this stage is merely optional.

In a third step, the variants obtained (mutants of the starting sequence) are screened for thiolase activity. For this purpose, the variants obtained are expressed in E. coli. The expression of the desired variant may be induced depending on the promoter as described in the prior art (eg Sambrook and Russell 2001). For a tetracycline-dependent system, expression can be induced by anhydrotetracycline as described in Example 2. After induction of expression, the cells are further grown and subsequently harvested, e.g. B. by centrifugation. Crude E. coli extracts expressing the thiolase variants are prepared as described in Example 3.

In one embodiment, the thiolase activity is tested in the absence of the inhibitor. In a further (preferred) embodiment, the thiolase activity is tested in the presence of the inhibitor. Merely optional, and to speed up the screening process, high-throughput screening is performed using multi-well plates, such as the 96-well microtiter plate format.

The thiolase activity measurements serve to identify improved thiolase variants. In a preferred embodiment, recombinant E. coli strains are used as host cells for the screening of a plasmid-derived library of the mutagenized thlA gene.

The determination of thiolase activities is performed by a 3-hydroxybutyryl-CoA dehydrogenase-coupled spectrophotometric assay as follows. The 3-hydroxybutyryl-CoA dehydrogenase activity is provided by a conventional 3-hydroxyacyl-CoA dehydrogenase or by the hbd protein of C. acetobutylicum or by the PhaB protein of R. eutropha. 3-hydroxybutyryl-CoA dehydrogenase activity is provided either by the purified enzymes or by crude extracts of native or recombinant host cells expressing the corresponding gene. The thiolase activities can be measured spectrophotometrically, for example in cuvettes or in microtiter plates (eg with 96 wells). In particular, the assay for determining thiolase activity according to the present invention is as follows:

Assay for the determination of thiolase activity:

Cell crude extracts of E. coli are prepared by adding 5 ml of BugBuster 10X protein extraction reagent (Merck Bioscience, Darmstadt, Germany) previously diluted 1:10 with 0.1 M Tris / HCl buffer, pH 6.0 , per gram of wet cell weight, and the suspension is incubated at room temperature for 20 min. To remove the cell debris, the suspension is centrifuged at 4 ° C and 13,000 rpm for 30 min. The supernatant is transferred to a fresh tube and subsequently used for enzyme activity measurements and the protein content is determined according to Bradford (Bradford 1976).

For the NADPH-coupled thiolase assay, acetoacetyl-CoA reductase (PhaB) activity is provided by crude extracts of E. coli BL21 (DE3) pET30 :: phaB1. [This crude extract is obtainable as follows: The phaB gene is amplified from the chromosomal DNA of R. eutropha H16 using the primers phaB_fw and phaB_rv and in pET-30 Xa / LIC according to the manufacturer's description (Merck Bioscience, Darmstadt, Germany , Cat # 69073-3). E. coli BL21 (DE3) pET30 :: pha8 is expressed in LB containing 100 μg / ml of ampicillin and 25 μg / ml chloramphenicol cultured at 37 ° C to an OD ₆₀₀ of 0.7, 24 mg / ml isopropyl-β-D-thiogalactopyranoside (IPTG) are added and the culture is incubated at 30 ° C for 4 hrs further incubated. The cells are harvested by centrifugation at 4 ° C and 5000 x g for 10 min and the cell pellets are stored at -20 ° C, if necessary. Cell crude extracts are prepared as described above.] The PhaB activity is measured spectrophotometrically: 30 μl of crude extract is added to 0.9 ml of potassium phosphate buffer (0.05 M, pH 6.0) containing 32 nM acetoacetyl-CoA and 100 nM NADPH , and absorbance is recorded at 340 nm (Haywood et al., 1988).

The condensation reaction of thiolase activity is assayed in extracts of E. coli BL21 (DE3) pASK-IBA3 :: thIA in 50 mM potassium phosphate buffer, pH 6.0, 1 mM dithiothreitol (DTT), 0.2 mM NADPH and 2 U PhaB (in Crude extracts of E. coli BL21 (DE3) pET30 :: phaBI). The reaction is started by adding 1 mM acetyl-CoA and monitored at 340 nm on an Ultrospec 3000 photometer (Amersham Buchler GmbH & Co. KG, Braunschweig, Germany). The reverse thiolytic cleavage reaction is measured spectrophotometrically by acetoacetyl-CoA decrease at 303 nm, as described by Hartmanis and Gatenbeck (1964).

Cell extract production from C. acetobutylicum strains and thiolase activity measurements are performed as previously described in detail (Hartmanis and Gatenbeck 1984, Lehmann and Lütke-Eversloh 2011). The above thiolase activity assay is performed to identify improved thiolase variants (mutants) among all the mutant thiolase variants obtained. Mutated thiolase variants can also be referred to as thlA ^MUT . As used herein, an improved thiolase mutant is a thiolase mutant (variant of the parental enzyme) that has either (a) higher activity than the parental enzyme (the parent enzyme of mutagenesis) (ie, without addition of CoA-SH) or (b) a higher activity than the parent enzyme in the presence of ≥ 10 μM CoA-SH or (c) a combination of (a) and (b) (ie both (a) and (b)). Improved thiolase variants can also be referred to as thlA ^IMP .

In a preferred embodiment of (a), an improved thiolase variant of the invention shows 5% or higher, preferably 10% or higher, more preferably 20% or higher, even more preferably 30% or higher activity compared to the parent (e.g. Wild-type) enzyme. When directed to the identification of enzymes of embodiment (a), no CoASH is added to the assay for determining activity.

In a preferred embodiment of (b), an improved thiolase variant has 50% or higher, preferably 75% or higher, more preferably 100% or higher, even more preferably 200% or higher activity in the presence of 10 μM CoA-SH in comparison to the parent (e.g., wild-type) enzyme. These include improved thiolase mutants that are not (measurably) inhibited by free CoA-SH. When directed to the identification of enzymes of embodiment (b), the 10 μM CoA-SH are added prior to performing the assay.

In the two preceding paragraphs, parenteral (eg, wild-type) enzyme refers to the polypeptide that served as the basis for the mutagenesis. The parent (e.g., wild-type) enzyme may be any enzyme possessing thiolase activity, such as any variant of ThlA, particularly any (preferably wild-type) ThlA from a Clostridium species. In a preferred embodiment, the parent (eg, wild type) enzyme is that shown as SEQ ID NO: 1.

The approach used by the inventors has also led to the identification of particularly preferred sites for mutagenesis, d. H. 133, 156, 222 with respect to SEQ ID NO: 1. It should be understood that the mutation of at least one, for example, any combination of two (ie, 133 and 156; 133 and 222 or 156 and 222) or all three of the Residues 133, 156, 222 may result in an improved enzyme of the invention. Thus, the present invention encompasses a polypeptide having thiolase activity characterized as being 70% or greater, preferably 75% or greater, more preferably 80% or greater, more preferably 85% or greater, more preferably 90% or greater preferably 95% or more, such as 96% or more, 97% or more, 98% or more, 99% or more sequence identity to SEQ ID NO: 1, provided that at least one, preferably any combination of two or all three of residues 133, 156, 222 have a mutation with respect to SEQ ID NO: 1. As used herein, the term "mutation" refers to any mutation selected from insertion, deletion and substitution, with substitution being preferred.

Preferred substitutions of position 133 are those wherein the radical R is represented by a small radical such as G or A or S or C, or a non-polar radical such as in particular a hydrophobic radical such as G, A, L, I, V, W, Y, F, with G being most preferred. Preferred substitutions of position 156 are those wherein the group H is replaced by an amide group such as N or Q, with N being preferred. Preferred substitutions of position 222 are those wherein the group G is replaced by a hydrophobic group, such as G, A, L, I, V, W, Y, F, with V being most preferred. Any combination of these preferred substitutions is also included in the invention. Thus, in the most preferred embodiment, the polypeptide is characterized in that the mutation (s) are selected from one, each combination of two and preferably all three of the following substitutions: R133G, H156N, G222V. The embodiment in which all these three substitutions are combined is shown in SEQ ID NO: 2 and is also referred to herein as thla ^OPT .

The genes of the improved thiolase variants can be expressed in an industrial production organism to increase the current into an acetoacetyl-CoA-dependent metabolic pathway. The DNA sequence of improved thiolase variants described in the present invention may be, e.g. By modifying codon usage, to the specific host organism for increased expression of the recombinant gene.

The improved thiolase enzymes can be used in a process for the preparation of industrially important compounds resulting from an acetoacetyl-CoA-related biosynthetic pathway.

More specifically, the improved thiolase enzymes may be involved in a process for producing an isoprenoid compound involving the mevalonate pathway. In a preferred embodiment, the improved thiolase enzymes for polyhydroxyalkanoate production by innate or recombinant hosts are used for the production of thermoplastic polyesters from renewable resources.

In another embodiment, improved thiolase enzymes are used to produce biofuels. In a preferred embodiment, a biofuel production process is based on the conversion of carbohydrates, such as mono-, di- or polysaccharides, optionally including lignocellulosic hydrolysates, into biofuels. The improved thiolase enzymes of the present invention are used in such a method.

The improved enzymes of the invention can be used in a process for producing a chemical along the acetoacetyl-CoA dependent pathway; this route requires thiolase activity. Any method using an enzyme of the invention is therefore also encompassed by the invention.

Preferably, the thiolase of the invention (used in the method of the invention) has 5% or higher, preferably 10% or higher, more preferably 20% or higher, even more preferably 30% or higher activity compared to the parental wild-type enzyme under physiological conditions. Physiological conditions are the conditions usually found in the organism in which the enzyme is expressed (optionally recombinantly). It is to be expected that a higher activity of the enzyme leads to a higher yield of the product of the corresponding route (eg butanol). The product yield can thus serve as an indirect measure of the activity. For direct measurement of activity, the enzyme is expressed as described above and the crude extract is assayed for thiolase activity as described above.

Preferably, the thiolase of the invention (to be used in the method of the invention) has an activity which is (substantially) not inhibited by 10 μM CoA-SH. This means that the activity in the presence of this concentration of CoA-SH corresponds to at least 90% of the activity in the absence of CoASH.

As described herein, this thiolase is obtainable by the screening methods described above for the selection of improved thiolase variants among the mutant thiolase variants. The screening may involve recombinant expression of the thiolase in bacteria, such as E. coli. The screening can be performed by spectrophotometric measurements of thiolase activities in bacterial cell crude extracts. Alternatively, the screening may involve the use of butyric acid for phenotype selection. The screening may include monitoring of polyhydroxyalkanoate accumulation and staining of viable colonies for phenotype selection.

Preferably, the chemical produced in the process of the invention is a C4 alcohol. C4 alcohol means any alcohol having four carbon atoms, and the number or type of non-H atoms is not particularly limited. Preferably, a C4 alcohol comprises an O atom as the sole non-H atom, i. H. is butanol (including n-butanol, 2-butanol and tert-butanol), preferably n-butanol.

The present invention also provides a nucleic acid encoding the improved enzyme of the invention. This includes the enzyme variant defined by the specific mutations as defined above, as well as any enzyme having a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A as defined above. In particular, the nucleic acid encoding the polypeptide defined by SEQ ID NO: 2 is included. It will be understood by those skilled in the art that due to the degeneracy of the genetic code, various nucleic acids can encode the polypeptide, and they are all included in the invention. The nucleic acids are best obtained by reverse translation in silico.

The nucleic acid may have a sequence of 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 96% or more (97% or more, 98% or more), and most preferably 99% or more sequence identity having SEQ ID NO: 13. In a particular embodiment, this level of sequence comprises the polynucleotide shown as SEQ ID NO: 13. SEQ ID NO: 13 encodes the polypeptide of SEQ ID NO: 2. In one embodiment, the sequence SEQ ID NO: 12 is excluded from the clouds of particular percent identities referred to in this paragraph; d. H. the invention also relates to a nucleic acid of 80% or more (including all of the above-listed more and most preferred embodiments) identity to SEQ ID NO: 13, provided that SEQ ID NO: 12 is excluded.

The nucleic acid may be in any form, although DNA (single-stranded or double-stranded, i.e., with the corresponding complementary strand) is preferred.

The invention further relates to a vector comprising the nucleic acid. Any suitable vector is encompassed by the invention, with circular nucleic acid vectors, in particular plasmids, being preferred. The vector will usually, although not necessarily, contain a promoter for the expression of the nucleic acid as defined above and / or an origin of replication enabling replication in the (or each of) the host cell (s) in which the vector is propagated and / or the nucleic acid defined above is to be expressed.

The invention further relates to a host cell comprising the above-defined nucleic acid or the vector defined above. The host cell is not particularly limited, although the criteria for its selection are based on the following considerations. It is preferred that the enzymes of the present invention be used for microbial butanol production. Organisms with pronounced C4 metabolism (preferably organisms capable of producing butanol) are a more preferred host for the expression of optimized thiolase genes according to the present invention. Thus, the host cell may be selected from organisms having a relatively high metabolic flux in a C4-specific pathway; these include in particular bacteria belonging to the genus Clostridium. In the method of the invention, the host cell may belong to the genus Clostridium. More preferably, the host cell belongs to one of the following species: C. acetobutylicum, C, beijerinckii, C. saccharoperbutylacetonicum, C. butyricum, C. pasteurianum, C. saccharobutylicum, C. Ijungdahlii, C. thermocellum, C. thermobutyricum, C. cellulolyticum ,

In other embodiments, the thiolase of the present invention may be expressed in another host organism, preferably unicellular organisms such as bacteria, archaea and unicellular fungi such as yeasts. In this case, additional genes encoding enzymes of the butanol biosynthetic pathway are optionally added (such as any combination of enzymes selected from the following list and preferably all of the following list: hydroxybutyryl-CoA dehydrogenease, crotonase, butyryl-CoA dehydrogenase, alcohol dehydrogenase ), (optionally recombinantly) expressed in the host organism. Thus, microorganisms suitable for butanol production include, but are not limited to, organisms that permit heterologous gene expression, such as Gram-negative bacteria (such as Escherichia coli) or Gram-positive bacteria (such as Bacillus subtilis) or even eukaryotic organisms (such as Saccharomyces cerevisiae). Thus, in this embodiment, butanol is not naturally butanol producing strains, ie a strain that does not naturally produce butanol. This includes more preferred embodiments in which the enzyme of the invention is expressed from a strain belonging to the genus Escherichia, Bacillus, Lactobacillus, Pseudomonas, Ralstonia, Saccharomyces, provided that the other enzymes for butanol production in the strain wherein the strain can be used for butanol production according to the invention. Preferably, the butanol is prepared from a strain belonging to the species E. coli, B. subtilis, L. brevis, L. plantarum, P. putida, P. fluorescens, P. aeruginosa, R. eutropha, S. cerevisiae.

The host cell of the invention optionally also expresses a gene encoding aldehyde / alcohol dehydrogenase, such as adhE1 (adh) from a Clostridium species, such as Clostridium acetobutylicum. The gene can be expressed homologously or heterologously. Overexpression is preferred.

In addition to in vivo metabolic pathways, improved thiolase can be used in vitro. For the in vitro synthesis of butanol, for example, the thiolase of the present invention as well as other enzymes of the butanol biosynthetic pathway (such as hydroxybutyryl-CoA dehydrogenease, crotonase, butyryl-CoA dehydrogenase, alcohol dehydrogenase) in vitro together with the reactants of the reaction and buffers provided.

Furthermore, the enzymes of the invention can be used in a synthetic route. A synthetic route can be designed in silico for the development of a process for the preparation of a desired compound which requires the formation of a carbon-carbon bond. In one embodiment, carbon-carbon bond formation occurs via thiolase-catalyzed acetoacetyl-CoA biosynthesis. This is particularly useful in embodiments in which the thiolase catalyzed reaction is the rate-limiting step of the synthetic route. Thus, thiolase enzymes as described in this invention can be used to achieve higher metabolic fluxes through the synthetic pathway due to increased thiolase activity and / or attenuated feedback inhibition.

EXAMPLES

Example 1: Bacterial strains and general culture conditions

E. coli was grown in LB medium containing per liter 5 g yeast extract, 10 g tryptone and 10 g NaCl, antibiotics to maintain the plasmid were added as needed (Sambrook and Russell 2001). C. acetobutylicum was anaerobically grown at 37 ° without shaking in Hungate tubes or serum bottles. Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) was added as a redox indicator for anaerobiosis at a concentration of 1 mg / l and residual oxygen was removed by adding 50-100 μl titanium (III ) nitrilotriacetic acid (NTA) solution (1.3 M NaOH, 0.16 M NTA, 0.27 M Na ₂ CO ₃ and 1.3% TiCl ₃ ). Fortified clostridial agar was used as the solid medium, 40 μg / ml of erythromycin was added to plasmid-containing strains of C. acetobutylicum. Procedures requiring strictly anaerobic conditions were performed in an anaerobic chamber with 90% N ₂ and 10% H ₂ . All strains used in this example are listed in Table I.

Table I. Bacterial strains, plasmids and oligonucleotides used in this study.

Example 2: Thiolase gene cloning and expression

General recombinant DNA techniques were performed according to standard protocols (Sambrook and Russell 2001). All cloning methods were validated by DNA sequencing (LGC Genomics GmbH, Berlin, Germany). The thlA gene (CAC2873) was amplified by PCR from chromosomal DNA of C. acetobutylicum ATCC 824 (Fischer et al., 2006) using the primers ThlA_EcoRI_fw and ThlA_ Kpnl_rv amplified and ligated via EcoRI and KpnI restriction sites in the vector pASK-IBA3, which gave the plasmid pASK-IBA3 :: thlA ^WT , which was transformed into E. coli BL21 (DE3). For protein expression, E. coli BL21 (DE3) pASK-IBA3 :: thlA ^WT and E. coli BL21 (DE3) pASK-IBA3 :: thlA ^MUT strains were mixed in LB medium with 100 μg / ml ampicillin at 37 ° C grown to an OD ₆₀₀ of 0.6-0.8. After addition of 20 μg / ml anhydrotetracycline (AHT), cultures were incubated overnight at 20 ° C and 180 rpm before crude extract preparation for enzyme activity assays.

The optimized thlA mutant gene identified in bank screening, SEQ ID NO: 2; thlA ^OPT , was subcloned into the vector pT via EcoRI / IKpnI restriction of pASK-IBA3 :: thlA ^OPT agarose gel purification, T4 DNA polymerase treatment and ligation, yielding the plasmid pT :: thlA ^OPT . After in vivo methylation in E. coli (Heap et al 2007; Mermelstein and Papoutsakis 1993), pT :: thlA ^{OPT was} electroporated into C. acetobutylicum ATCC 824 as previously described (Riebe et al., 2009). All plasmids and oligonucleotides used in this example are listed in Table I.

Example 3: Enzyme activity measurements

Cell crude extracts of E. coli were prepared by adding 5 ml of BugBuster 10X protein extraction reagent (Merck Bioscience, Darmstadt, Germany), previously diluted 1:10 with 0.1 M Tris / HCl buffer, pH 6.0, per Grams of wet cell weight and the suspension was incubated at room temperature for 20 minutes. To remove the cell debris, the suspension was centrifuged at 4 ° C and 13,000 rpm for 30 min. The supernatant was used for measurements of enzyme activity and the protein content was determined according to Bradford (Bradford 1976).

For the NADPH-coupled thiolase assay, acetoacetyl-CoA reductase (PhaB) activity was provided by crude extracts of E. coli BL21 (DE3) pET30 :: phaB1. For this, the phaB gene from chromosomal DNA of R. eutropha H16 was amplified using the primers phaB_fw and phaB_rv and amplified in pET-30 Xa / LIC according to the manufacturer's description (Merck Bioscience, Darmstadt, Germany, cat. 3) cloned. E. coli BL21 (DE3) pET30 :: phaBI was cultured in LB containing 100 μg / ml ampicillin and 25 μg / ml chloramphenicol at 37 ° C to an OD ₆₀₀ of 0.6-0.8, 24 mg / ml of isopropyl-β-D-thiogalactopyranoside (IPTG) were added and the culture was further incubated at 30 ° C for 4 hrs. The cells were harvested by centrifugation at 4 ° C and 5000 x g for 10 min, and the cell pellets were stored at -20 ° C, if necessary. Cell crude extracts were prepared as described above. The PhaB activity was measured spectrophotometrically: 10-50 μl of crude extract was added to 0.9 ml of potassium phosphate buffer (0.05 M, pH 6.0) containing 32 nM acetoacetyl-CoA and 100 nM NADPH, and absorption was monitored 340 nm recorded (Haywood et al., 1988).

The condensation reaction of thiolase activities has been reported in extracts of E. coli BL21 (DE3) pASK-IBA3 :: thlA in 50 mM potassium phosphate buffer, pH 6.0, containing 1 mM dithiothreitol (DTT), 0.2 mM NADPH and 2 U PhaB (in crude extracts of E. coli BL21 (DE3 ) pET30 :: phaB1). The reaction was started by addition of 1 mM acetyl-CoA and monitored at 340 nm on an Ultrospec 3000 photometer (Amersham Buchler GmbH & Co. KG, Braunschweig, Germany). The reverse thiolytic cleavage reaction was measured spectrophotometrically by acetoacetyl-CoA decrease at 303 nm as described (Hartmanis and Gatenbeck (1984). Cell extract preparation from C. acetobutylicum strains and thiolase activity measurements were performed as previously described in detail (Hartmanis and Gatenbeck 1984 Lehmann and Lütke-Eversloh 2011). Enzyme activity measurements in E. coli cell extracts are shown in Table II. Enzyme activities in cell extracts of E. coli hosts. Acetoacetyl-CoA reductase (PhaB) Thiolase (ThlA) tribe NADH NADPH Thiolytic cleavage condensation E coli BL21 (DE3) pASK-IBA3 (control) 0.03 ± 0.02 0.02 ± 0.01 0.08 ± 0.04 0.2 ± 0.09 E. coli BL21 (DE3) pASK-IBA3 :: thlA ^WT 0.05 ± 0.03 0.73 ± 0.09 65.94 ± 4.61 68.96 ± 1.54 E. coli tuner (DE3) pET30 :: phaB1 0.15 ± 0.02 34.27 ± 1.43 0.02 ± 0.003 0.3 ± 0.11

Example 4: Thiolase Bank Construction

To construct the thlA mutant library (termed pASK-IBA3 :: thlA ^MUT ), synthetic oligonucleotides that formed the degenerate backbone of the library were assembled without any involved amplification reaction (non-amplified library) and the diversity of the library was directly related to the amount correlated to produced DNA molecules, ie 67 fmol / ul corresponded to 4 × 1010 molecules (Geneart AG, Regensburg, Germany). To prepare the amplified library, 4 μl (268 fmol or 1.6 x 10 ¹¹ molecules) of the unamplified library were amplified with primers T7_fw and M13_rv, full length fragments were gel purified and resuspended in 100 μl Tris / EDTA buffer. The concentration of the amplified library was determined by UV spectroscopy to be 400 ng / μl. The amplified library was cleaved with EcoRI and KpnI and ligated into the EcoRI / KpnI digested vector pASK-IBA3. Ligation reactions were transformed into E. coli BL21 (DE3) and the transformation rate was determined by plating out serial dilutions. The number of transformants was 5.7 × 10 ⁴ colony forming units (CFU). Total cells from the transformation plates were harvested for plasmid preparation. The concentration of the cloned library was determined to be 0.46 μg / μl by UV spectroscopy. The plasmid preparations were sequenced with the primers pASK_fw, pASK_rv and 1107435.S1_rv (Geneart AG, Regensburg, Germany). Total cells from the transformation plates were harvested, resuspended in 50% by volume glycerol and aliquots of the cell suspension, which included 3.9 x 10 ¹⁰ cells / ml, were stored at -70 ° C. All strains, plasmids and oligonucleotides used in this example are listed in Table I.

Example 5: Screening method

The E. coli pASK-IBA3 :: thlA ^WT library was plated on LB agar plates plus 100 μg / ml ampicillin (LB + Ap) and incubated overnight at 37 ° C. Colonies were transferred with sterile toothpicks into 96-well microtiter plates containing 200 μl LB + Ap and grown overnight at 37 ° C. Each microtiter plate each comprised two samples of E. coli pASK-IBA3 :: thlA ^WT and E. coli pASK-IBA3 (empty vector) as controls. 10 μl aliquots of the cultures were used to inoculate fresh LB + Ap and the grown microtiter plates were stored at -70 ° C after addition of 10% by volume glycerol. The inoculated microtiter plates were incubated at 37 ° C until the cultures showed an OD ₆₀₀ of 0.5-0.7. For the induction of gene expression, 20 μg / ml AHT was added and the cultures were incubated overnight at 20 ° C. Subsequently, crude cell extracts were prepared using BugBuster 10X Protein Extraction Reagent (Merck Bioscience, Darmstadt, Germany) as described above to determine the thiolase activities of the E. coli pASK-IBA3 :: thlA ^MUT library. For this, 150 μl of potassium phosphate buffer (50 mM, pH 6.0) containing 1 mM DTT and 0.2 mM NADPH were pipetted into the wells of fresh microtiter plates. In the analysis of CoA-SH sensitivity, the buffer contained 10 μM free coenzyme A (Sigma-Aldrich Chemie, Deisenhofen, Germany). After addition of 20 .mu.l of PhaB extract (2 U) and 10 .mu.l of cell crude extracts, the reaction was started by adding 20 .mu.l of acetyl-CDA solution (20 mM). Absorbance at 340 nm was recorded for 3 min at 3 sec intervals on a SpectraMax M2e multi-mode microtiter plate reader and the slope values were calculated automatically.

To validate the results of the microtiter plate screening, the 14 positive clones were grown on a larger scale and the thiolase activities were determined spectrophotometrically in the cuvette format (Table III). None of the ThlA ^MUT variants showed increased total thiolase activity, but differences to ThlA ^WT were detected in the presence of 10-50 μM CoA-SH. While 8 clones showed activities similar to wild-type ThlA at different CoA-SH concentrations, clones 9G4, 13D7, 19C10, 21B8, 25C8 and 28C9 had significantly increased thiolase activities when CoA-SH was added to the assay (Table III ). Subsequent DNA sequencing of the respective plasmids revealed that all 6 positive clones had exactly the same genotype, suggesting that in fact only one new ThlA ^MUT variant had been isolated. In essence, the 6 independently identified hits confirmed the reliability of the screening method for finding improved thiolase mutants. Thus, clone 9G4 was chosen as a proxy for subsequent experiments and was designated as E. coli BL21 (DE3) pASK-IBA3 :: thlA ^OPT . Table III. Thiolase activities of putative positive clones identified in the screening of the E. coli BL21 (DE3) pASK-IBA3 :: thlA ^MUT library. ThIA variant Thiolase activity in the presence of free CoA-SH 0 μM 10 μM 20 μM 30 μM 40 μM 50 μM wildtype 68.96 ± 1.54 25.80 ± 0.84 8.44 ± 0.67 1.30 ± 0.03 0.28 ± 0.03 0.11 ± 0.03 7D5 69.80 ± 3.41 26.23 ± 1.58 8.54 ± 0.13 1.33 ± 0.08 0.29 ± 0.04 0.13 ± 0.01 9G4 69.19 ± 0.55 34.60 ± 1.67 17.23 ± 1.06 9.83 ± 0.25 2.06 ± 0.10 1.18 ± 0.08 11b9 69.39 ± 2.01 24.81 ± 0.58 8.38 ± 0.06 1.22 ± 0.04 0.30 ± 0.05 0.10 ± 0.03 12B8 69.50 ± 1.37 25.23 ± 0.23 8.52 ± 0.09 1.30 ± 0.12 0.27 ± 0.03 0.11 ± 0.03 13D7 68.94 ± 0.89 36.45 ± 0.68 17.28 ± 0.57 11.87 ± 0.08 1.98 ± 0.17 1.17 ± 0.07 17C5 70.11 ± 1.27 24.58 ± 0.77 8.30 ± 0.41 1.06 ± 0.04 0.32 ± 0.02 0.13 ± 0.02 19C10 69.76 ± 0.90 37.89 ± 0.53 18.57 ± 0.58 10.52 ± 0.28 1.86 ± 0.76 1.43 ± 0.09 21B8 68.72 ± 0.12 34.13 ± 1.06 16.79 ± 0.43 11.32 ± 0.20 2.37 ± 0.09 1.38 ± 0.15 22B7 69.85 ± 1.19 24.85 ± 0.98 8.47 ± 0.09 3.76 ± 4.83 0.32 ± 0.05 0.13 ± 0.02 25C8 70.33 ± 0.53 35.92 ± 0.47 17.33 ± 0.53 9.95 ± 0.77 2.12 ± 0.18 1.34 ± 0.19 28B9 68.61 ± 0.54 24.80 ± 0.53 8.90 ± 0.73 1.02 ± 0.08 0.37 ± 0.02 0.15 ± 0.01 28C4 69.88 ± 1.00 24.61 ± 0.81 8.42 ± 0.30 1.12 ± 0.03 0.31 ± 0.05 0.14 ± 0.00 28C9 69.51 ± 1.00 37.89 ± 0.53 20.73 ± 4.51 9.83 ± 0.33 2.00 ± 0.17 0.01 ± 1.23 30F3 71.03 ± 0.79 24.56 ± 0.65 8.41 ± 0.01 1.32 ± 0.20 0.29 ± 0.04 0.12 ± 0.01

Example 5: Fermentation experiments for solvent production

Cultivation experiments of recombinant C. acetobutylicum were performed in 200 ml of MS-MES containing 40 μg / ml of erythromycin, as previously described in detail (Lehmann et al 2012a, Lehmann and Lütke-Eversloh 2011, Lehmann et al., 2012b). , Samples were taken regularly to determine the pH with a pH meter (Wissenschaftlich-Technische Werkstätten GmbH, Weilheim, Germany) and the optical density at 600 nm in a spectrophotometer (Zeiss Spekol 1100-Photometer, Analytik Jena AG, Jena, Germany ) using 1.5 ml plastic cuvettes with a 1 cm light path. Samples of cell-free supernatant were stored at -20 ° C until quantification of glucose (Bergmeyer 1983) and fermentation products. Acetate, butyrate, acetone, ethanol and butanol were prepared according to Thormann et al. (Thormann et al., 2002) on an Agilent 7890A gas chromatograph (Agilent Technologies, Boeblingen, Germany) equipped with a Chromosorb 101 (80/100 mesh, 2.0 m x 3.0 mm x 1.6 mm) glass column was equipped, certainly.

When the protein represented as SEQ ID NO: 2 was recombinantly expressed in C. acetobutylicum, the following results were obtained: In addition to significantly delayed ethanol and acetone formation, the ethanol titer was increased by 46%. The butanol titer was increased by 18%, whereas the final acetone concentrations were similar to the vector control strain.

Protein sequences shown in the Sequence Listing

• SEQ ID NO: 1: Thl wild type (Clostridium acetobutylicum)
• SEQ ID NO: 2: ThlOPT
• SEQ ID NO: 12: Nucleic acid encoding the polypeptide of SEQ ID NO: 1
• SEQ ID NO: 13: Nucleic acid encoding the polypeptide of SEQ ID NO: 2

Other oligonucleotide sequences shown in the sequence listing are also listed in Table I.

LITERATURE SERVICES REFERRED TO IN THE DESCRIPTION

PATENT DOCUMENTS REFERRED TO IN THE DESCRIPTION

• US 4,757,010 Production of clostridium acetobutylicum mutants of high butanol and acetone productivity, the resultant mutants and the use of these mutants in the joint production of butanol and acetone
• US 6,358,717 Method of producing butanol using a mutant strain of Clostridium beijerinckii
• US 2011/0027845 A1 , Enhanced ethanol and butanol using the same
• US020110281313 A1 , Improved production of acid and solvent in microorganisms
• CN 000101423813 B Recombinant clostridium and construction method and use thereof

NON-PATENT LITERATURE REFERRED TO IN THE DESCRIPTION

• Dürre P. 1998. New insights and novel developments in clostridial acetone / butanol / isopropanol fermentation. Appl Microbiol Biotechnol 49: 639-648.
• Drought P. 2011. Fermentative production of butanol - the academic perspective. Curr Opin Biotechnol 22: 331-336.
• Fischer RJ, Oehmcke S, Meyer U, Mix M, Schwarz K, Fiedler T, Bahl H. 2006. Transcription of the pst operon of Clostridium acetobutylicum is dependent on phosphate concentration and pH. J Bacteriol 188: 5469-5478.
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Sequence List.txt SEQUENCE LOG

Claims (16)

A method of producing a chemical using an acetoacetyl-CoA dependent pathway having a thiolase activity with higher specific activity and / or reduced sensitivity to the inhibitor coenzyme A compared to the activity and sensitivity of the polypeptide of SEQ ID NO: 1, wherein the thiolase is a mutant protein having at least one mutation in the CoA-SH binding loop domain relative to the parental wild-type enzyme, and wherein the determination of thiolase activity is performed by a 3-hydroxybutyryl-CoA dehydrogenase-coupled spectrophotometric assay. The method of claim 1, wherein the thiolase has 5% or higher, preferably 10% or higher, more preferably 20% or higher, even more preferably 30% or higher activity compared to the parental wild-type enzyme under physiological conditions. Method according to one of claims 1 or 2, wherein the activity of the thiolase is not inhibited by 10 μM CoA-SH. The method of any of claims 1-3, wherein the chemical is a C4 alcohol. The method of claim 4, wherein the chemical is butanol, preferably n-butanol. The method of any of claims 1-5, wherein the production is in a host cell belonging to the genus Clostridium. The method of claim 6, wherein such a host cell belongs to one of the following species: C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum, C. butyricum, C. pasteurianum, C. saccharobutylicum, C. Ijungdahlii, C. thermocellum, C. thermobutyricum, C. cellulolyticum. A process according to any one of claims 5-7, wherein butanol is produced by non-natural butanol producing strains. A method according to any one of claims 5-8, wherein butanol is produced by a strain belonging to the genus Escherichia, Bacillus, Lactobacillus, Pseudomonas, Ralstonia, Saccharomyces. A method according to any of claims 5-9, wherein butanol is produced by a strain belonging to the species E. coli, B. subtilis, L. brevis, L. plantarum, P. putida, P. fluorescens, P. aeruginosa, R. eutropha, S. cerevisiae. A polypeptide having thiolase activity, characterized in that it is 70% or more, preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, such as 96% % or greater, 97% or greater, 98% or greater, 99% or greater sequence identity to SEQ ID NO: 1, provided that at least one, such as any combination of two or all three of residues 133, 156 , 222 have a mutation (selected from substitution, deletion or insertion) with respect to SEQ ID NO: 1. The polypeptide of claim 11, wherein the mutation (s) is selected from • any, • any combination of two and Preferably all three (SEQ ID NO: 2) of the following substitutions: R133G, H156N, G222V is / are. Method according to one of claims 1 to 10, characterized in that the thiolase activity is provided by the enzyme according to any one of claims 11 to 12. A nucleic acid encoding a polypeptide selected from the group consisting of (a) the polypeptide of any one of claims 11 or 12 and (b) the enzyme having the thiolase activity as defined in any one of claims 1 to 10. A vector comprising the nucleic acid of claim 14. A host cell harboring the nucleic acid of claim 14 or the vector of claim 15.

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