(19)
(11)EP 3 080 285 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
27.07.2022 Bulletin 2022/30

(21)Application number: 14824634.1

(22)Date of filing:  10.12.2014
(51)International Patent Classification (IPC): 
C12N 9/88(2006.01)
C12P 7/24(2006.01)
C12N 9/10(2006.01)
C12P 7/26(2006.01)
C12N 15/52(2006.01)
C12N 9/04(2006.01)
C12P 7/40(2006.01)
(52)Cooperative Patent Classification (CPC):
C12P 7/24; C12P 7/26; C12P 7/40; C12N 15/52; C12N 9/0006; C12N 9/1025; C12N 9/88; C12Y 101/01085; C12Y 203/03013; C12Y 402/01033
(86)International application number:
PCT/US2014/069438
(87)International publication number:
WO 2015/089127 (18.06.2015 Gazette  2015/24)

(54)

PROCESSES TO PREPARE ELONGATED 2-KETOACIDS AND C6-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

VERFAHREN ZUR HERSTELLUNG LÄNGLICHER 2-KETOSÄUREN UND C6-C10-VERBINDUNGEN DARAUS ÜBER GENETISCHE MODIFIKATIONEN AN DEN MIKROBIELLE STOFFWECHSELWEGEN

PROCÉDÉS DE PRÉPARATION DE 2-CÉTOACIDES ALLONGÉS ET DE COMPOSÉS EN C6-C10 À PARTIR DE CEUX-CI PAR MODIFICATIONS GÉNÉTIQUES VERS DES VOIES MÉTABOLIQUES MICROBIENNES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 12.12.2013 US 201361915040 P

(43)Date of publication of application:
19.10.2016 Bulletin 2016/42

(73)Proprietor: Dow Global Technologies LLC
Midland, MI 48674 (US)

(72)Inventors:
  • SANGHANI, Paresh C.
    Indianapolis, IN 46268 (US)
  • STOWERS, Christopher C.
    Indianapolis, IN 46268 (US)
  • RODRIGUEZ, Brandon A.
    Freeport, TX 77541 (US)
  • VENKATESWARAN, Amudhan
    Indianapolis, IN 46268 (US)

(74)Representative: Boult Wade Tennant LLP 
Salisbury Square House 8 Salisbury Square
London EC4Y 8AP
London EC4Y 8AP (GB)


(56)References cited: : 
WO-A2-2010/045629
  
  • RYAN J MARCHESCHI ET AL: "A Synthetic Recursive +1 Pathway for Carbon Chain Elongation", ACS CHEMICAL BIOLOGY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US, vol. 7, no. 4, 1 January 2012 (2012-01-01) , pages 689-697, XP008162820, ISSN: 1554-8929, DOI: 10.1021/CB200313E [retrieved on 2012-07-13]
  • E A FELNAGLE ET AL: "Engineering synthetic recursive pathways to generate non-natural small molecules", NATURE CHEMICAL BIOLOGY, vol. 8, 17 May 2012 (2012-05-17), pages 518-525, XP055173427, ISSN: 1552-4450, DOI: 10.1038/nchembio.959
  • SHEN C R ET AL: "A SYNTHETIC ITERATIVE PATHWAY FOR KETOACID ELONGATION", METHODS IN ENZYMOLOGY, VOL 497: SYNTHETIC BIOLOGY, METHODS FOR PART/DEVICE CHARACTERIZATION AND CHASSIS ENGINEERING, PT A ELSEVIER ACADEMIC PRESS INC, 525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495 USA SERIES : METHODS IN ENZYMOLOGY (ISSN 0076-68, vol. 497, 2011, pages 469-481, XP009182941,
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The invention relates to the field of using biological enzymes to produce C7-C11 2-ketoacids and further C6-C10 aldehydes anc products made therefrom. More particularly, it relates to the field of using engineered enzymes that can be either expressed by a genetically-modified microbial organism to convert a suitable substrate to a C6-C10 aldehyde via one or more metabolic pathways, or can be incubated in vitro to convert 2-ketobutyrate or 2-ketoisovalerate to an elongated 2-ketoacid and then to convert the 2-ketoacid to a C6-C10 aldehyde.

[0002] Concerns about the future scarcity, cost, and environmental impact of obtaining and using fossil fuels have stimulated interest in the exploitation of cheap, renewable biomass as alternative sources for both fuels and chemicals made from them. As crude oil prices have risen, biobased chemicals and industrial products have become attractive alternatives to their petroleum-derived counterparts. Fermentation processes using anaerobic microbial organisms offer a promising path for converting biomass and agricultural wastes into useful products, while at the same time remediating problems that may be encountered in disposal of low-value agricultural commodities and food processing byproducts/wastes. Some of the useful products that can be prepared from low-cost biomass feedstocks are organic acids and alcohols, including in particular C6-C10 alcohols such as octanol. Octanol finds particular use as a lower-cost starting material to prepare octene, which is a highly desirable feedstock chemical in a number of industries. These industries include the polyethylene industry, which uses octene as a co-monomer for solution polymerizations, and the detergent industry, which uses it to alkylate phenols to produce detergent precursors. Octene has been typically prepared by linear alpha olefin (LAO) technology which relies on energy-intensive tetramerization, and as a result, octene supplies are often insufficient, contributing to undesirable fluctuations in its price. Thus, identification of better and less expensive methods to produce octanol would be expected to lead to less expensive production of octene. In general, use of fermentation processes to produce octanol could provide such cost reduction, which would in turn increase supply and stabilize the price of octene.

[0003] In general methods for the improvement of industrial microbial organisms range from the random approach of classical strain improvement (CSI) to the highly rational methods of metabolic engineering. CSI is often effective for alleviating product inhibition or improving productivity, but is a far less effective approach to generate strains capable of producing entirely new products. Furthermore, CSI is intensive as to both time and resources. This is because, in order to obtain strains with high tolerance to inhibitory fermentation products, it is necessary to continuously screen and select mutants by successively culturing the strain in the media in the presence of increasing inhibitor concentrations. This is usually carried out in conjunction with induced mutagenesis using chemical mutagens and/or ultraviolet (UV) radiation. However, the conventional culture screening process is generally tedious, time-consuming, and often fruitless.

[0004] Metabolic modifications are frequently more effective at creating strains that produce new products. This is because genes, and in some cases even entire pathways, can be transferred between organisms (recombinant methods), and/or enzymes can be modified (engineered methods). These methods avoid some of the disadvantages of CSI. Metabolic engineering, a term comprehending both recombinant and engineered methods, is a targeted and often faster approach that is widely used to design strains to achieve higher efficiencies in metabolite overproduction, through alterations in the metabolic flux distribution. Most of this work to date is related to the production of secondary metabolites (such as antibiotics), amino acids (e.g., lysine), and heterologous proteins, using organisms with well-studied genetics and physiology (e.g., E. coli, yeast, and hybridoma cells). Stoichiometric analysis of metabolic flux distributions provides a guide to appropriate metabolic modification, optimal medium formulation and feeding strategies, and bioprocess optimization. However, this approach still requires in-depth knowledge of the metabolic and regulatory networks in the fermentation cells. Although these rational approaches have been successful in cases involving a single gene or a few genes within a single gene cluster, they have often been ineffective in cases involving more complex or largely unknown metabolic pathways. This is because such approaches usually target one gene at a time, and thus fail to predict complex interactions among multiple genes in a given pathway.

[0005] Enzyme modification is performed by modifying that portion of the genetic code, i.e., the organism's DNA, which corresponds to the expression of that enzyme. Modification of enzymes can lead to entirely new functionality or may be used to improve the specificity or efficiency of desired intermediates or products. Additionally, certain enzymes are known to be promiscuous, often performing tasks beyond their known natural roles. Such enzymes may also be modified to perform novel conversions, but to date the success of this approach has frequently been limited to product yields that are not commercially viable. See, e.g., Zhang, K.; Sawaya, M.R.; Eisenberg, D.S.; Liao, J.C. "Expanding metabolism for biosynthesis of nonnatural alcohols," Proc. Natl. Acad. Sci. USA, 2008, 105:20653-20658. Modifying multiple enzymes in a pathway may theoretically be used as a technique to maximize specificity and/or catalytic efficiency.

[0006] One example of an organism known to produce octanol under certain conditions is Clostridium. Various species of Clostridium (e.g., C. acetobutylicum, difficile, and kluyveri) are employed in WO2012135731. That publication ascribes a Clostridium species' poorly selective production, that includes a small amount of n-octanol, among other products, to the engineered microbial organism's ability to express or overexpress beta-ketothiolase (e.g., BktB), acetyl CoA acetyltransferase (e.g., AtoB), 3-hydroxybutyryl-CoA dehydrogenase (e.g., Hbd, from the Clostridium, or PaaHI), crotonase (e.g., Crt), and trans-enoyl-CoA reductase (e.g., Ter). These modifications are generally to the organism's CoA pathway for the production of higher alcohols. This pathway avoids the butanol production pathway found in many species of Clostridium, which involves oxygen-sensitive enzymes and intermediates. However, the amount of n-octanol produced via this invention is too small to be commercially viable. See also, e.g., Lee, J.Y.; Jang, Y.S.; Lee, J.; Papoutsakis, E.T.; Lee, S.Y. "Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production," Biotechnol., 2009, 4:1432-1440; and Wang, Y.; Blaschek, H.P. "Optimization of butanol production from tropical maize stalk juice by fermentation with Clostridium beijerinckii," Bioresour. Technol., 2011, 102, 9985-9990.

[0007] WO2010045629 discloses the same enzymatic pathway with LeuABCD for carbon chain elongation and ketoacid production wherein mutations in LeuA were disclosed to enlarge substrate specificity or to reduce feedback resistance by exchanging amino acids in the active site for the process of a catalytic cycle to elongate ketoacids.

[0008] Described herein is a process for preparing a C7-C11 2-ketoacid comprising contacting a substrate, selected from 2-ketobutyrate and 2-ketoisovalerate, and (1) a native or genetically modified LeuA enzyme; (2) a genetically modified LeuB' enzyme, wherein the enzyme is (a) obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof; or (b) the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (a); and (3) a native or genetically modified LeuCD' enzyme complex; under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid.

[0009] The invention provides a process for preparing a C7-C11 2-ketoacid comprising contacting a substrate, selected from 2-ketobutyrate and 2-ketoisovalerate, and

(1) a native or genetically modified LeuA enzyme having isopropylmalate synthase activity;

(2) a genetically modified LeuB' enzyme, wherein:

(a) the enzyme is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified (1) at Leu-96 by substituting glycine; (2) at Val-198 by substituting alanine; (3) at Leu-96 by substituting alanine and at Val-198 by substituting alanine; (4) at Leu-96 by substituting glycine and at Val-198 by substituting alanine; (5) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or (6) at Leu-96 by substituting alanine;

or (b) the enzyme has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (a);

and (3) a native or genetically modified LeuCD' enzyme complex having isopropylmalate isomerase activity; under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid.



[0010] In another embodiment, the C7-C11 2-ketoacid produced as described in the previous paragraph is contacted with a native or genetically modified thiamin dependent decarboxylase, under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted.

[0011] In still another embodiment, the invention provides a microbial organism that expresses a genetically modified LeuB' enzyme that:

(1) is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified (i) at Leu-96 by substituting glycine; (ii) at Val-198 by substituting alanine; (iii) at Leu-96 by substituting alanine and at Val-198 by substituting alanine; (iv) at Leu-96 by substituting glycine and at Val-198 by substituting alanine; (v) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or(vi) at Leu-96 by substituting alanine;

or (2) has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, and wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1, the enzyme having been modified as in (1).



[0012] Also described herein is a microbial organism that expresses or overexpresses an enzyme that is (1) obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof; or (2) has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (1).

[0013] In yet another embodiment the invention provides a process to prepare a C6-C10 aldehyde comprising

(1a) contacting a carbon-containing substrate and: native or genetically modified pyruvate carboxylase having pyruvate carboxylase activity, native or genetically modified aspartate aminotransferase having aspartate aminotransferase activity, native or genetically modified bifunctional aspartokinase/homoserine dehydrogenase having bifunctional aspartokinase/homoserine dehydrogenase activity, native or genetically modified homoserine kinase having homoserine kinase activity, native or genetically modified threonine synthase having threonine synthase activity, native or genetically modified aspartate semialdehyde dehydrogenase having aspartate semialdehyde dehydrogenase activity and native or genetically modified threonine dehydratase having threonine dehydratase activity, under conditions to form 2-ketobutyrate, or

(1b) contacting a carbon containing substrate and: native or genetically modified acetolactate synthase isozyme with acetolactate synthase isozyme activity, ketol-acid reductoisomerase having ketol-acid reductoisomerase activity, and dihydroxy-acid dehydratase having dihydroxy-acid dehydratase activity, under conditions to form 2-ketoisovalerate;

(2) contacting the 2-ketobutyrate or 2-ketoisovalerate and

  1. (a) a native or genetically modified LeuA enzyme having isopropylmalate synthase activity;
  2. (b) a genetically modified LeuB' enzyme, wherein the LeuB' enzyme

    (i) is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified (a) at Leu-96 by substituting glycine; (b) at Val-198 by substituting alanine; (c) at Leu-96 by substituting alanine and at Val-198 by substituting alanine; (d) at Leu-96 by substituting glycine and at Val-198 by substituting alanine; (e) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or (f) at Leu-96 by substituting alanine;

    or (ii) has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, and wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (i); and

  3. (c) a native or genetically modified LeuCD' enzyme complex having isopropylmalate isomerase activity; under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid; and

(3) contacting the C7-C11 2-ketoacid and a native or genetically modified thiamin dependent decarboxylase having thiamin dependent decarboxylase activity under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted;

each of (1), (2) and (3) occurring in one or more steps;

wherein (1), (2) and (3) independently occur within or outside of a genetically modified microbial organism.



[0014] Also described herein is a process to prepare a C6-C10 aldehyde comprising (1) contacting a carbon-containing substrate and one or more native or genetically modified enzymes under conditions to form 2-ketobutyrate or 2-ketoisovalerate; (2) contacting the 2-keto-butyrate or 2-ketoisovalerate and (a) a native or genetically modified LeuA enzyme; (b) a genetically modified LeuB' enzyme, wherein the enzyme is (i) obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified in that alanine, glycine, valine or leucine is independently substituted for Leu-96, Val-198, or a combination thereof; or (ii) the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (a); and (c) a native or genetically modified LeuCD' enzyme complex; under conditions such that the 2-ketobutyrate or 2-keto-isovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid; and (3) contacting the C7-C11 2-ketoacid and a native or genetically modified thiamin dependent decarboxylase under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted; each of (1), (2) and (3) occurring in one or more steps; wherein (1), (2) and (3) independently occur within or outside of a genetically modified microbial organism.

FIGURE 1 shows the elongation of a 2-ketoacid by the recursive activities of LeuABCD, in steps 1 to 3. Following the elongation, the resulting elongated 2-ketoacid (IV) is then converted to an aldehyde (V), via the activity of a (thiamin dependent) decarboxylase in step 4, and finally to an alcohol (VI), via the activity of an alcohol dehydrogenase in step 5.

FIGURE 2 shows two related but different routes to produce 1-octanol. In one, a Wood-Ljungdahl pathway converts synthesis gas to acetyl CoA, and another pathway then converts the acetyl CoA to pyruvate. The pyruvate is then converted to 2-ketobutyrate, and finally a LeuABCD pathway is initiated, wherein the 2-ketobutyrate is converted to (in this embodiment) 2-ketononanoate. Once the elongated 2-ketoacid has been formed (the 2-ketononanoate), a (thiamin dependent) decarboxylase converts it to a C6-C10 aldehyde (in this embodiment, octanal), and an alcohol dehydrogenase converts the octanal to a C6-C10 alcohol (in this embodiment, 1-octanol).



[0015] In the other route, also illustrated in FIGURE 2, one of the potential sugar catabolism pathways, which in this embodiment is a glycolysis or pentose phosphate pathway, converts a C5 or C6 sugar to pyruvate, and thereafter the same pathway sequence is followed as in the first route to reach the 1-octanol.

[0016] SEQUENCE LISTING, SEQ ID 1, shows a native (wild type) E. coli LeuB gene sequence, with both base pairs and the corresponding amino acids. Sequence Listing Free Text states as follows: "Wild type E. coli LeuB."

[0017] SEQUENCE LISTING, SEQ ID 2, show the amino acids for the native (wild type) E. coli LeuB, but without the corresponding base pairs.

[0018] SEQUENCE LISTINGS, SEQ ID 3-20, show LeuB' variant enzymes with specific base pair modifications (shown in odd-numbered sequences) and amino acid modifications (without corresponding base pairs) (shown in even-numbered sequences), as described. Sequence Listing Free Text (<223>) for each states the designation of the particular variant. The designations are defined and discussed in the text hereinafter.

[0019] In general the present invention includes a process to convert 2-ketobutyrate or 2-keto-isovalerate to a C7-C11 2-ketoacid; a genetically modified enzyme or combination of enzymes to carry out that conversion; and a process to prepare a C6-C10 product, which may be an aldehyde, an alcohol, a carboxylic acid, or an alkane, via contact between a carbon-containing substrate, such as, for example, synthesis gas (syngas) or a C5 or C6 sugar, such as sucrose, glucose, or pentose, and a series of enzymes that convert the carbon-containing substrate to the C6-C10 product via one or more steps, and in certain embodiments via three or more steps, and preferably five or more steps. Either process may be carried out biosynthetically, in one of the described embodiments of a non-naturally occurring, i.e., genetically engineered, cell, i.e., in a non-naturally occurring microbial organism; or via in vitro methodology.

[0020] In the process to prepare a C6-C10 aldehyde, a selected carbon-containing substrate is converted first to pyruvate, and from pyruvate to either 2-ketobutyrate or, alternatively, to 2-ketoisovalerate, via the action of one or more enzymes and in one or more steps. The 2-ketobutyrate or 2-ketoisovalerate is then converted, via chain elongation, to a C7-C11 2-ketoacid, by means of the action of at least one of three non-native, i.e., genetically modified, enzymes or enzyme complexes or a combination thereof in the LeuABCD pathway (as it is termed with respect to the E. coli microbial organism), which is a portion of the non-natural leucine pathway (FIGURE 1). The first group of potentially employed modified enzymes accomplishing this chain elongation are identified herein as constituting Leu A' (2-isopropylmalate synthase), Leu B' (isopropylmalate dehydrogenase), and Leu CD' (two enzymes that, together, are termed isopropylmalate isomerase complex). The C7-C11 2-ketoacid may then be converted to a C6-C10 aldehyde by the action of at least one more enzyme, including a native or genetically modified thiamin dependent decarboxylase, which converts the C7-C11 2-ketoacid to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted.

[0021] Finally, the C6-C10 aldehyde may be used as is, in a variety of industrial applications, or may be employed as an intermediate or starting material for production of other chemicals. For example, the C6-C10 aldehyde may be contacted with an alcohol dehydrogenase, which converts the C6-C10 aldehyde to the corresponding C6-C10 alcohol. Alternatively, it may be contacted with an aldehyde dehydrogenase, which converts it to the corresponding C6-C10 carboxylic acid. Finally, it may be contacted with a fatty aldehyde decarbonylase, which converts it to the corresponding C5-C9 alkane. Each of the thiamin dependent decarboxylase and the alcohol dehydrogenase, aldehyde dehydrogenase, or fatty aldehyde decarbonylase may independently be native or genetically modified.

[0022] The terms "genetically modified," or "modified," as used herein, refers to an enzyme (whether generally or a specifically named enzyme, e.g., alcohol dehydrogenase, etc.) having an intentionally altered amino acid sequence, or a microbial organism (depending upon placement of either term as an adjective) having an intentionally altered genome. Such alteration may have been accomplished via recombinant technology, where one or more genes are transferred from a second, different microbial organism into a target microbial organism; engineered technology, wherein the amino acids within the target microbial organism are altered, generally via site-directed mutagenesis, resulting in the conversion of at least one amino acid (and frequently more than one) to a different amino acid; or both.

[0023] In preferred embodiments the C6-C10 product, for example, a C6-C10 alcohol such as 1-octanol, is produced with desirably high specificity, i.e., preferably at least 25 percent (%), more preferably at least 40 %, still more preferably at least 50 %, and most preferably at least 70 %, based on weight (wt) of total product (i.e., wt%), is the targeted product. Another way of stating this is that the yield of desired product (e.g., 1-octanol) is preferably at least 0.25 gram product per gram of feedstock (g/g), more preferably at least 0.4 g/g, still more preferably at least 0.5 g/g, and most preferably at least 0.7 g/g.

[0024] As noted hereinabove, the invention may be carried out either in vivo or in vitro. An in vivo approach may be preferred for commercial scale production, while an in vitro approach may be more convenient for laboratory and general research purposes, such as to carry out enzymatic assays. In general the in vivo approach employs a microbial organism's native metabolic pathway(s), first to convert a suitable carbon-containing substrate to pyruvate, and then to convert the pyruvate to 2-ketobutyrate or, alternatively, to 2-ketoisovalerate, in a varying number of steps.

[0025] In one embodiment, the selected microbial organism may possess a Wood-Ljungdahl pathway, also known as a "synthesis (syngas) fixation pathway," wherein syngas is converted to acetyl CoA. Such may be carried out effectively by certain acetate-producing bacteria species, suchas those of the genus Clostridium, including but not limited to, in particular, Clostridium Ijungdahlii (C. Ijungdahlii). In this pathway, conversion of the syngas to acetyl CoA generally includes reduction of carbon dioxide to carbon monoxide, and then to acetyl CoA via the action of two enzymes, i.e., carbon monoxide dehydrogenase, which catalyzes the reduction of the carbon dioxide, and acetyl CoA synthase, which combines the resulting carbon monoxide with a methyl group to form acetyl CoA. From this point the acetyl CoA continues on another pathway wherein it is converted to pyruvate, via reduction by PFOR (ferrodoxin oxidoreductase). Such pathways may be present in organisms including, for example, Clostridium, Escherichia coli (E. coli), Azospirillum, Bacillus, Saccharomyces and Corynebacterium.

[0026] In an alternative embodiment, a suitable (non-syngas) carbon-containing substrate, such as a C5 or C6 sugar (glucose, sucrose, pentose, or a combination thereof), may be converted directly to pyruvate via one of the sugar catabolism pathways, such as a glycolysis or pentose phosphate pathway.

[0027] Thereafter the pyruvate may be converted first to L-threonine, via PC (pyruvate carboxylase); AAT (aspartate aminotransferase); ThrABC (ThrA, which is a bifunctional aspartokinase/homoserine dehydrogenase); ThrB, which is homoserine kinase; and ThrC, which is threonine synthase); and ASD (aspartate semialdehyde dehydrogenase). The L-threonine is then converted to 2-ketobutyrate via ILva (threonine dehydratase). In an alternative embodiment, the pyruvate may be converted to 2-ketoisovalerate via the activities of IlvBN/IlvGM, IlvC, and IlvD. See, also, Zhang, K.; Sawaya, M.R.; et al., ibid.

[0028] Following production of 2-ketobutyrate or 2-ketoisovalerate, genetic modification of the native LeuABCD portion of the non-natural leucine biosynthesis pathway operates to effect conversion to a C7-C11 2-ketoacid via one or more steps. In certain embodiments several steps are involved and employ, in an in vivo approach, at least one modified (endogenous or exogenous) enzyme, enzyme complex, or combination thereof (collectively referred to herein as "LeuA'B'CD‴), to convert 2-ketobutyrate or 2-ketoisovalerate to a desired C7-C11 2-ketoacid. For example, 2-ketobutyrate is converted first to 2-ketovalerate, then to 2-ketocaproate, then to 2-ketoheptanoate or up to 2-keto-undecanoate, depending upon the desired final product, as chain-lengthening occurs. Alternatively, 2-ketoisovalerate is converted first to 2-ketoisocaproate, then to 2-ketoisoheptanoate, and so forth. However, it is optionally possible to modify only one enzyme, enzyme complex or combination thereof-for example, LeuA', LeuB', or LeuCD' - to obtain acceptable or desirable production of a C7-C11 2-ketoacid beginning with 2-ketobutyrate or 2-ketoisovalerate.

[0029] Once an elongated C7-C11 2-ketoacid is formed, such may be used as is, or converted to a C6-C10 aldehyde. For such conversion, a native or genetically modified thiamin dependent decarboxylase (DC') is employed, resulting in a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted. C6-C10 aldehydes enjoy wide applicability both in themselves and as starting or intermediates in producing C6-C10 alcohols, carboxylic acids, alkanes, and combinations thereof, as described hereinabove. Production of a C6-C10 alcohol, in one embodiment, octanol, is illustrated in FIGURE 1.

[0030] In order to enable a non-native organism to carry out some portion of the conversions in vivo as defined hereinabove, for example, to produce the C6-C10 aldehydes and/or C6-C10 alcohols, it is desirable to perform protocols similar to that described hereunder. In general the Examples included herewith involve enzyme engineering to alter the amino acids in order to modify enzyme functionality, particularly in terms of activity and/or specificity. This alteration in the amino acids may be used to produce modified enzyme for small scale purposes, for example, for in vitro assays; or may be the basis for genome modification in order to produce a strain of microbial organisms suitable for larger scale production.

[0031] The methodology may be carried out as is well understood by those skilled in the art. In general, a suitable database, such as GenBank, is used to obtain the genetic codes for the native enzyme(s), followed by identification of the codons suitable for modification. This identification may be used as the basis for art-known methods of protein engineering, wherein computer molecular modeling identifies and also enables differentiation of structural locations at which modifications of enzyme/substrate interfaces may be effectively employed. A given desirable modification is then performed, using a molecular biology technique called site-directed mutagenesis. The modified gene is then cloned into a replicative plasmid vector which, when transformed into a host microbial organism such as E. coli, enables the production of enzymes having a higher-than-native catalytic efficiency. The E. coli cells containing the targeted variant enzyme also produce other native proteins and, therefore, the variant-type enzymes must then be subjected to purification to separate out non-targeted proteins and general cell structures, leaving a purified enzyme that will exhibit a higher-than-native, i.e., higher than wild type, catalytic efficiency. This can be appropriately assayed in vitro, according to the methodology most suited to the given particular enzyme. An assayed enzyme that is shown to have a desirable level of catalytic efficiency is thereby confirmed to be the product of a desirable genetic modification, and may be used for in vitro production methods, such as for the in vitro production of a given C7-C11 2-ketoacid, such as 2-ketononanoate, and/or a C6-C10 aldehyde, such as octanal, and/or a product made from the C6-C10 aldehyde, such as a C6-C10 alcohol, carboxylic acid, or alkane.

[0032] A particular application for the above-described methodology is to produce a desirable organism for large or otherwise commercial scale fermentative production of an enzyme-facilitated product, such as a C6-C10 aldehyde or one of the C6-C10 products that may be prepared therefrom. Such preparation may be carried out by inserting the DNA, or pieces of DNA, which encode for the desired improved enzyme into the genome of a second microbial organism known or believed to possess other desirable characteristics, such as, for example, capability to resist inhibitors during fermentation, capability to produce pyruvate (or acetyl CoA) from a particular carbon-containing substrate, or some other advantageous trait(s). Thus, the second microbial organism is now genetically-modified, in that it produces a genetically modified enzyme.

[0033] In another embodiment, it is also possible to simply identify a microbial organism having native enzymes that are useful in a desired overall pathway, and either use that microbial organism itself as a starting microbial organism, or transfer the appropriate enzyme-encoding portion of the genome(s) of such microbial organism(s) into the genome of the organism that has been already identified as being useful for large scale fermentation production. An example of this would be to select a microbial organism that already produces a suitable native thiamin dependent decarboxylase (DC) and native alcohol dehydrogenase (ADH), and use that microbial organism either as a starting organism or as a transformant organism to prepare a genetically modified microbial organism to produce a C6-C10 alcohol at higher yields or specificity than wild type microbial organisms.

Example 1


Preparing the modified LeuA (i.e., "LeuA‴) enzyme.



[0034] This example represents an embodiment wherein an engineered LeuA' enzyme is prepared. Such is accomplished beginning with an Escherichia coli organism that has been transformed with a plasmid containing a modified LeuA gene to produce a 2-isopropylmalate synthase variant (LeuA') having a higher-than-average catalytic efficiency (kcat/KM) for capturing 2-ketoacids of interest for catalysis. For this a particular gene that has been identified as being suitable is LeuA; GenBank Accession No. NC_000913.3 Gene ID: 947465.

[0035] In the expression kcat/KM, kcat is the "turnover number," of unit = sec-1; KM is the Michaelis-Menten constant; and the turnover number equals VMax/[E], wherein VMax is maximum velocity, and [E] is the enzyme concentration. The equation applies to reactions obeying Michaelis-Menten kinetics, and generally provides the amount of substrate, in moles, that is converted to product in one second. The value kcat/KM therefore indicates an effective catalysis capability and is obtained experimentally. The variants having a relatively higher kcat/KM are therefore those that are more efficient at condensing 2-ketoacids (n = 1-5 in intermediate I in FIGURE 1), which may (in this exemplary FIGURE 1) include 2-ketobutyrate, 2-ketovalerate, 2-ketocaproate, 2-ketoheptanoate, and/or 2-ketooctanoate, with acetyl CoA, thereby generating the corresponding 2-alkylmalate products (intermediate II in FIGURE 1).

[0036] To prepare LeuA' variants substitutions are made at amino acid residue sites designated as Phe-47, Leu-73, His-97, Phe-99, Ser-139, and Asn-167. One or more of these targeted amino acids is/are converted to the amino acids glycine, alanine, leucine or valine by performing site-directed mutagenesis of the known LeuA gene of a selected organism, such as E. coli (GenBank: Accession No. NC_000913.3 Gene ID: 947465). See, also, U.S. Patent 8,298,798 B2. A histidine "tag" (histidine-tag) may be attached to any given protein as an aid in purification of the protein. Following this "tagging," which may comprise a varying length sequence inserted at either end of a selected sequence and may include six (6) histidines and selected additional amino acids, the histidine-tagged enzyme, which constitutes a variant of the enzyme (i.e., a modified enzyme) (Leu A') which is expressed in the E. coli DE3 cells, is purified via nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The efficiency of the purified variant enzyme in condensing the various 2-ketoacids (n = 1-5 in intermediate I in FIGURE 1) is determined by in vitro enzyme assays. For more information on one assay that may be suitable for this determination, see, e.g., Marcheschi, R.J.; Li, H.; Zhang, K.; Noey, E.L.; Kim, S.; Chaubey, A.; Houk, K.N.; Liao, J.C. "Synthetic recursive "+1" pathway for carbon chain elongation," ACS Chem. Biol., 2012, 7:689-697. Variants displaying higher levels of catalytic efficiency (kcat/KM) are identified and selected for production of one or more desired C6-C10 alcohol(s). Combinations of variants of LeuA' that work together to catalyze the condensation of any or all of the identified 2-ketoacids involved in the C6-C10 alcohol biosynthesis may also be identified on the basis of in vitro assays.

[0037] FIGURE 2 shows two routes to form 2-ketobutyrate from a carbon-containing substrate. The first is via a combination of a Wood-Ljungdahl syngas fixation pathway to form pyruvate, followed by a pyruvate to 2-ketobutyrate pathway, and the second is via a combination of a glycolysis or pentose phosphate pathway to form pyruvate, followed by a pyruvate to 2-ketobutyrate pathway. In either case, once 2-ketobutyrate is formed, the LeuABCD pathway begins chain elongation to produce the desired C7-C11 2-ketoacid (in this embodiment, 2-ketononanoate), and this C7-C11 2-ketoacid is then decarboxylated via a (thiamin dependent) decarboxylase to form the C6-C10 aldehyde having one less carbon than the C7-C11 2-ketoacid being decarboxylated (in this embodiment, octanal). Finally, the C6-C10 aldehyde is acted upon by an alcohol dehydrogenase to produce the corresponding C6-C10 alcohol (in this embodiment, 1-octanol).

Example 2


I. Preparing E. coli LeuB' (isopropylmalate dehydrogenase) variants having increased activity against 3-hexylmalate (3-HM).



[0038] During 2-ketononanoate biosynthesis by the recursive activity of the LeuABCD pathway, 3-alkylmalic acids of varying lengths are formed as substrates of LeuB. For efficient biosynthesis of 2-ketononanoate, it is desired that LeuB efficiently capture 3-ethylmalate (intermediate III, n=2; FIGURE 1), 3-propylmalate (Intermediate III, n=3; FIGURE 1), 3-butylmalate (3-BM; Intermediate III, n=4; FIGURE 1), 3-pentylmalate (Intermediate III, n=5; FIGURE 1) and 3-hexylmalate (3-HM; Intermediate III, n=6; FIGURE 1) for catalysis. The native LeuB is relatively inefficient in capturing longer nonnatural 3-alkylmalate substrates. To improve the efficiency of native LeuB in capturing 3-hexylmalate for catalysis, the active site of native LeuB is modified using protein engineering techniques as described hereinbelow.

[0039] Residues lining the 3-isopropylmalate binding site of E. coli LeuB were identified from a structural model of LeuB that is constructed via homology modeling and using as the template the crystal structure model of Thiobacillus ferrooxidans isopropylmalate dehydrogenase (Protein Data Bank (PDB) code 1A05), as reported by Imada, K.; Inagaki, K.; Matsunami, H.; Kawaguchi, H.; Tanaka, H.; Tanaka, N.; Namba, K. "Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 Å resolution: the role of Glu88 in the unique substrate-recognition mechanism," Structure, 1998, 6:971-982. Leu-96 and Val-198 are selected for modification and variants are prepared wherein one or both is/are replaced, variously, with valine, alanine, and/or glycine, as shown in Table 1.
Table 1
EnzymeNucleotide sequence in the LeuB gene at indicated positions
Wild type LeuB 95-Leu-Leu-Pro-97 283-CTGCTGCCT -291 197-Asn -Val -Leu-199 589-AACGTGCTG-597
L96A 283-CTGGCCCCT-291 589-AACGTGCTG-597
L96V 283-CTGGTGCCT-291 589-AACGTGCTG-597
L96G 283-CTGGGTCCT-291 589-AACGTGCTG-597
V198A 283-CTGCTGCCT-291 589-AACGCGCTG-597
V198G 283-CTGCTGCCT-291 589-AACGGTCTG-597
L96A/V198A 283-CTGGCCCCT-291 589-AACGCCCTG-597
L96G/V198A 283-CTGGGTCCT-291 589-AACGCGCTG-597
L96G/V198G 283-CTGGGTCCT-291 589-AACGGTCTG-597
L96V/V198G 283-CTGGTGCCT-291 589-AACGGTCTG-597
Enzymes are identified by the modifications made to the wild type amino acid sequence. The name of each includes a first letter which is the abbreviation for the wild type enzyme's amino acid; the number is its position within the amino acid sequence; and the last letter, which is the abbreviation for the amino acid substituted at that location. L = leucine; A = alanine; G = glycine; and V = valine.


[0040] For example, enzyme L96A is prepared by replacing Leu-96 in E. coli isopropylmalate dehydrogenase with alanine; enzyme L96G is prepared by replacing Leu-96 with glycine; and enzyme L96G/V198A is prepared by replacing Leu-96 with glycine and Val-198 with alanine. The remaining LeuB variants in the Table 1 are named according to the amino acid replaced.

[0041] Each of the engineered LeuB variants is expressed, purified, and then evaluated for activity against three substrates, which are 3-isopropylmalate (3-IPM), 3-butylmalate (3-BM), and 3-hexylmalate (3-HM). 3-IPM is the natural substrate of LeuB and is formed in the microbial organisms during the biosynthesis of leucine. The 3-BM and 3-HM are non-natural substrates of LeuB that would be formed inside the cells during C7-C11 2-ketoacid, for example, 2-ketononanoate, biosynthesis.

[0042] The evaluation of the LeuB variants is performed in two steps using the high-throughput enzyme assay described below. Initially, all the variants are tested for activity against a single high concentration of 3-IPM, 3-BM and 3-HM. Following the initial evaluation, detailed kinetic analysis is performed on a select number of variants to determine the maximal rate (kcat), Michaelis-Menten constant (KM), and the catalytic efficiency of the enzyme (kcat/KM).

[0043] LeuB' variants that are more efficient (higher kcat/KM) than the wild type enzyme in converting all or some of the 3-alkylmalate substrates, such as 3-HM, to the corresponding C7-C11 2-ketoacid, are desirable because they improve the overall efficiency of the relevant "+1" pathway. II. Heterologous expression of LeuB (isopropylmalate dehydrogenase) and its engineered variants in E. coli.

[0044] To evaluate the substrate specificity of the wild type LeuB and the engineered LeuB' variants listed in Table 1, genes of all the proteins are expressed into E. coli cells separately and the protein products isolated from the cells. To obtain the protein products, the LeuB gene sequence (EcoGene Accession No. EG11577 (Sequence Listing, SEQ ID 1) is downloaded from the EcoGene website (http://ecogene.org). Codons of an additional 11 amino acids are added after the codon of the last amino acid in the open reading frame of the LeuB gene. These additional amino acids include 6 histidines plus 5 additional amino acids, and are attached as a histidine-tag, to aid in purifying the protein in a single step using Ni-NTA chromatography. The gene sequence of the entire LeuB sequence with the 11 additional amino acids is chemically synthesized, cloned into the pETDuet-1 vector (EMD Biosciences), downstream of the T7 polymerase promoter, and sequenced by Synthetic Genomics Inc. (San Diego, CA). It is noted that none of the Sequence Listings included herein show the histidine-tag that is used, which in this case is Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser.

[0045] Genes of the LeuB' variants listed in Table 1 are also chemically synthesized following replacement of the codon of the selected amino acid (Leu-96 and/or V-198) with that of alanine, valine and/or glycine (Sequence Listing, SEQ ID 2 through SEQ ID 10) and cloned into the pETDuet-1 vector. The pETDuet-1 vector containing the LeuB or its variant gene is transformed into E. coli, and the LeuB or its variant is expressed and eventually purified as described below.
  1. A. Transformation of E. coli: E. coli expression studies are conducted using the competent BL21 (DE3) cells acquired from EMD Biosciences. Transformations are performed as per the kit instructions and involve mixing a 50 microliter (µL) aliquot of competent cells with 1 µL of the vector. Cells harboring the LeuB expression vector are selected using ampicillin as the marker.
  2. B. Expression of LeuB and its variants in E. coli: E. coli transformants harboring the expression vector of LeuB or its variant are selected on LB agar plates containing 100 micrograms per milliliter (µg/mL) of ampicillin. The plates are incubated at 37 degrees Celsius (°C) for 16 hours (h). A starter culture is started with transferring a single colony of transformant into 50 milliliters (mL) of Lysogeny Broth (LB) medium containing 100 µg/mL of ampicillin and incubated at 37 °C with shaking at 220 revolutions per minute (rpm) for overnight. Next day, 7 mL of starter culture is inoculated into 800 mL of Terrific Broth (TB) and the culture is incubated at 37 °C until the culture reaches an optical density at 600 nanometers (nm) (OD600nm) of 0.5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 millimolar (mM) is added to induce the expression of the LeuB or LeuB variant (LeuB') genes, and the culture is transferred to a 15 °C incubator for 16 h. At the end of 16 h, the culture is centrifuged at 8000 rpm to pelletize the cells. The cell pellet is divided into two aliquots and stored at -80 °C overnight before purification.
  3. C. Purification of LeuB and its variants: An E. coli cell pellet from 400 mL of expression culture is suspended in B-PER reagent (Thermo Fisher Scientific, Inc., Rockford, IL) containing 1 µg/mL of DNAse (Thermo Fisher Scientific, Inc., Rockford, IL), 1 µg/mL of lysozyme (Thermo Fisher Scientific, Inc., Rockford, IL), 1 mM dithiothreitol (DTT), and protease inhibitor cocktail (RPI Corp., Mount Prospect, IL). The suspension is rocked gently for 30 minutes (min) at room temperature and centrifuged at 15,000 times gravity (x g) for 20 min to pelletize the cell debris. The supernatant is separated and incubated with 5 mL of Co-NTA resin (Thermo Fisher Scientific, Inc., Rockford, IL) that has been pre-equilibrated with an equilibration buffer (50 mM sodium phosphate, pH 8.0, containing 300 mM sodium chloride, 20 mM imidazole, 50 µL protease inhibitor cocktail, and 15 percent by volume (vol%) glycerol). Following an incubation period of 1 h at 4 °C, the LeuB bound resin is washed with 5 volumes of equilibration buffer. LeuB and its variants are then eluted from the Co-NTA resin with an equilibration buffer containing 200 mM imidazole. The eluted proteins are finally dialyzed against phosphate buffered saline and stored as a 20 vol% glycerol solution at -20 °C.

III. Determination of the substrate specificity of the wild type and the engineered LeuB variants.



[0046] The evaluation of the LeuB variants is performed in two steps using the high-throughput enzyme assay described below. Initially, all of the variants are screened for activity against a single high concentration of 3-IPM, 3-BM and 3-HM. Following the initial evaluation, detailed kinetic analysis is performed on a select number of variants to determine the maximal rate (kcat), Michaelis-Menten constant (KM), and the catalytic efficiency of the variant enzymes (kcat / KM).

[0047] A suitable spectrophotometric LeuB enzyme assay is adapted into a high-throughput format in 96-well plates for the kinetic evaluation of LeuB and its variants (shown in Table 1) against 3-isopropylmalate (3-IPM), 3-butylmalate (3-BM), and 3-hexylmalate (3-HM). See, for example, Hsu, Y.; Kohlhaw, G.B. "Leucine biosynthesis in Saccharomyces cerevisiae. Purification and characterization of beta-isopropylmalate dehydrogenase," J. Biol. Chem., 1980, 255:7255-7260. Steady state kinetic parameters (kcat, KM and kcat/KM) for the oxidative decarboxylation of the three substrates by the variants are determined in these experiments.
  1. A. HTP screening assay for the identification of functional LeuB variants: The HTP screening assay involves incubating 750 micromoles (µM) of 3-IPM, 1 mM 3-BM or 1 mM 3-HM, with 2 mM nicotinamide adenine dinucleotide (NAD+) in LeuB assay buffer (50 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), pH 8.0, containing 30 mM potassium chloride (KCI) and 5 mM magnesium chloride (MgCl2)) at 25 °C. The reaction is started by the addition at 25 °C of working enzyme stock containing from 0.3 microgram (µg) to 1.1 µg of LeuB variant diluted in assay buffer containing 1 mM DTT and 1 milligram per milliliter (mg/mL) bovine serum albumin (BSA). The plate containing the 200 µL of reaction mixture is centrifuged at 2500 x g for 15 seconds (sec) and the absorbance change of the reaction mixture is followed spectrophotometrically at 340 nm on a BioTek plate reader, pre-equilibrated at 25 °C. Initial velocity of the enzyme reaction is calculated from the rate of NADH production at 340 nm and using the extinction coefficient of NADH (6.22 per millimolar per centimeter (mM-1cm-1)). The activity of all the variants is normalized with the amount of enzyme present in the reaction mixture and expressed as nanomoles per minute per microgram (nmol.min-1.µg-1) (Table 2). Protein concentrations for normalizing the activities are determined using the 660 nm total protein assay kit acquired from Pierce Biotechnology Inc., available from Thermo Fisher Scientific, Inc., and using BSA as the standard.
  2. B. HTP LeuB enzyme assay for the determination of the kinetic parameters of oxidative decarboxylation of 3-IPM, 3-BM and 3-HM by LeuB variants:
    The HTP kinetic assay involves incubating eight varied concentrations, of 3-IPM (from 0 to 1.2 mM) or 3-BM (from 0 to 2 mM) or 3-HM (from 0 to 2 mM), with 2 mM NAD+ in LeuB assay buffer (50 mM HEPES, pH 8.0, containing 30 mM KCl and 5 mM MgCl2) at 25 °C. The reaction is started by adding from 0.008 µg to 0.5 µg of LeuB variant which has been pre-diluted in assay buffer containing 1 mM DTT and 1 mg/mL BSA. The plate containing the 200 µL of reaction mixture is centrifuged at 2500 x g for 15 sec and the absorbance change is followed spectrophotometrically at 340 nm on a BioTek plate reader maintained at 25 °C. Initial velocity of the enzyme reaction is calculated from the rate of NADH production at 340 nm and using the extinction coefficient of NADH (6.22 mM-1cm-1). The kinetic parameters (kcat, KM and kcat/KM) of oxidative decarboxylation of 3-alkylmalate are determined by fitting the data to the Michaelis-Menten equation using nonlinear regression performed using the GraphPad Prism software. Table 3 lists the kinetic parameters of the wild type and engineered variants against the three substrates. The amount of enzyme in the reaction mixture is determined using the 660 nm total protein assay kit acquired from Pierce Biotechnology Inc., using BSA as the standard.
  3. C. Results and Discussion


[0048] To improve the efficiency of the "+1" pathway in producing 2-ketononanoate, the isopropylmalate dehydrogenase would desirably efficiently catalyze the oxidative decarboxylation of all of the intermediate 3-alkylmalates. The three substrates used for the evaluation of LeuB variants are representative of these intermediate 3-alkylmalates, i.e., 3-isopropylmalate (3-IPM) is representative of the shorter 3-alkylmalate substrates expected to form during the earlier cycles of the "+1" iterative pathway; 3-butylmalate (3-BM) is representative of the intermediate-length 3-alkylmalates; and formation; and 3-hexylmalate (3-HM) would be oxidatively decarboxylated by LeuB to form 2-ketononanoate.

[0049] Initially the LeuB' variants are screened for activity against a single high concentration of each of 3-IPM, 3-BM and 3-HM (Table 2).
Table 2. Evaluation of the Wild type LeuB and LeuB' variants.
EnzymeSpecific activity, nmol.min-1.ug-1
3-IPM3-BM3-HM
Wild type LeuB 18.3 4.3 0.68
L96A 15.4 9.48 0.77
L96V 1.53 ND ND
L96G 2.90 1.5 9.5
V198A 2.83 1.81 2.5
V198G 4.66 1.83 0.5
L96A/V198A 0.65 1.26 3.3
L96G/V198A ND 1.49 20.4
L96G/V198G 0.02 0.08 3.6
L96V/V198G 0.05 0.04 ND
The specific activity of LeuB variants against the three substrates, 3-isopropylmalate (3-IPM), 3-butylmalate (3-BM), and 3-hexylmalate (3-HM) is determined as described in the HTP screening assay. The specific activities shown are the means of duplicated experiments from the screening assay.
The range between the two values is less than 20 %. ND=not detected under experimental conditions.


[0050] Without wishing to be bound by any theory, the results illustrated in Table 2 may be interpreted as suggesting that replacing Leu-96 and/or Val-198 with amino acids having smaller hydrophobic side chains, e.g., valine, alanine, or glycine, may in some instances simultaneously decrease enzyme activity against 3-IPM, and increase enzyme activity against 3-HM.

[0051] Variants L96A, L96G, V198A, L96A/V198A, L96G/V198A, and L96G/V198G exhibit higher activity than the wild type enzyme against 3-HMe and are subjected to further kinetic analysis. This analysis suggests that the wild type LeuB is highly efficient in capturing its native substrate, 3-IPM for catalysis. As illustrated in Table 3, the catalytic efficiency of the wild type enzyme is found to be 1,000-fold higher than that of the 3-HM, and the efficiency of capturing 3-BM is intermediate of the two substrates. The wild type LeuB enzyme thus becomes a progressively poorer catalyst as the "+1" pathway iterates for elongating 2-ketobutyrate to a C7-C11 2-ketoacid, such as, in this instance, 2-ketononanoate.

[0052] Substituting Leu-96 in the E. coli LeuB with progressively smaller residues, e.g., alanine (L96A) or glycine (L96G), results in improving the efficiency of capturing substrates with longer alkyl chains, with a concomitant reduction in capturing substrates with shorter alkyl chains. The L96A variant is approximately 4-fold more inefficient at capturing 3-IPM than the wild type enzyme, while being modestly poor in capturing 3-BM (see Table 3). The L96G variant is 100-fold less efficient than the wild type enzyme in capturing 3-IPM, while being 80-fold more efficient at capturing 3-HM than the wild type enzyme. This suggests that Leu-96 is blocking the capture of longer alkylmalates, such as 3-HM.

[0053] Substituting Val-198 in the E. coli LeuB with a smaller amino acid residue, e.g., alanine, improves the catalytic efficiency of the enzyme in capturing longer 3-alkylmalates, such as 3-HM, while decreasing the efficiency of capturing shorter alkylmalates, such as 3-IPM. This is evident from the comparative efficiencies of the V198A variant in catalyzing the conversion of 3-HM, which is 5-fold higher, and of 3-IPM, which is 1,000-fold lower, when compared to the efficiencies of the wild type LeuB (see Table 3).
Table 3. Substrate specificity of engineered LeuB enzymes
EnzymeSubstratekcat min-1KM mMKcat/KM, Min-1.mM-1
Wild typeLeuB 3-IPM 1435 ± 121 0.009 ± .003 167,805 ± 39,622
  3-BM 219 ± 20 0.038 ± 0.013 6274 ± 1778
  3-HM 26 ± 2 0.16 ± 0.02 166 ± 16
L96A 3-IPM 666 ± 154 0.016 ± 0.01 45,447 ± 13,020
  3-BM 420 ± 22 0.13 ± 0.02 3315 ± 372
  3-HM 41 ± 0.5 0.24 ± 0.04 179 ± 33
L96A/V198A 3-IPM * * 36 ± 12
  3-BM * * 67 ± 6
  3-HM 159 ± 4 0.08 ± 0.01 2008 ± 287
V198A 3-IPM * * 151 ± 71
  3-BM * * 94 ± 26
  3-HM 97 ± 33 0.11 ± 0.01 898 ± 333
L96G 3-IPM * * 178 ± 41
  3-BM 187 ± 6 1.9 ± 0.2 94 ± 6
  3-HM 455 ± 10 0.03 ± 0.003 13,159 ± 754
L96G/V198A 3-IPM * * 60 ± 9
  3-BM 268 ± 21 3.3 ± 0.4 79 ± 3
  3-HM 874 ± 23 0.05 ± 0.0005 16,932 ± 217
3-IPM = 3-isopropylmalate; 3-BM = 3-butylmalate; and 3-HM = 3-hexylmalate. For LeuB' variants having high Michaelis-Menten constant (KM) against one of the substrates, only the catalytic efficiency (kcat/KM) is reported. Mean ± standard deviation (S.D.) from a minimum of three independent experiments is shown.
Indicates that activity is low and, therefore, the parameter cannot be calculated.


[0054] It is also noted that substitution of both Leu-96 and Val-98 with smaller hydrophobic residues simultaneously increase in efficiency of capturing 3-HM and decreases the efficiency of capturing 3-IPM. The L96A/V198A and L96G/V198A variants exhibit a 12- and 102-fold greater efficiency, respectively, in capturing 3-HM compared with the wild type enzyme (Table 3), while at the same time these variants are very poor catalysts for 3-IPM, as evident from a drop of more than 3000-fold when compared with the wild type LeuB enzyme.

[0055] The data shows that the genetically modified LeuB' enzyme is generally operates at a higher catalytic efficiency than that of the wild type enzyme to catalyze, as shown, 3-butylmalate to form 2-ketoheptanoate, or 3-hexylmalate to form 2-ketononanoate. It can also be inferred that it will more efficiency catalyze 3-pentylmalate to form 2-ketooctanoate. Finally, it will also carry out combinations of these conversions at a higher catalytic efficiency.

[0056] Kinetic data on LeuB' variants suggests that one means of efficient formation of a C7-C11 2-ketoacid, such as but not limited to 2-ketononanoate, by the iteration of the "+1" pathway would be realized by coexpression of the wild type LeuB enzyme and one or more of the variants listed in Table 3.

Example 3


Preparing the modified LeuCD (i.e., "LeuCD' ") enzymes.



[0057] In this Example 3 embodiment an approach similar to that used for the LeuA' enzyme in Example 1 is described. The process begins with an E. coli organism that has been transformed with a plasmid containing the LeuC and LeuD genes so that it produces a modified 2-isopropylmalate isomerase complex with higher catalytic efficiency (kcat/KM) at isomerizing 2-alkylmalates (n = 1-5 in Intermediate II, FIGURE 1) generated by LeuA' to their corresponding 3-alkylmalates (n = 1-5 in Intermediate III, FIGURE 1). The modified LeuC and LeuD genes are made synthetically by altering some of the genetic codes, i.e., substituting one or more of the amino acids in the amino acid sequence, of LeuC and LeuD genes obtained from GenBank (LeuC: GenBank Accession No. NC 000913.3 Gene ID: 945076; and LeuD: GenBank Accession No. NC 000913.3, Gene ID: 945642). Each engineered variant is made by replacing one or more residues within and/or near the active sites of LeuC and/or LeuD using site-directed mutagenesis. Each modified enzyme is expressed as a histidine-tagged protein in E. coli DE3 cells and then purified using Ni-NTA chromatography. The efficiency of the purified variant enzyme in isomerizing various 2-alkylmalates is determined by an in vitro coupled enzyme assay such as may be used to assay for aconitase. See, e.g., Han, D.; Canali, R.; Garcia, J.; Aguilera, R.; Gallaher, T.K.; Cadenas, E. "Sites and mechanisms of aconitase inactivation by peroxynitrite: Modulation by citrate and glutathione," Biochemistry, 2005, 44:11986-11996. Variants displaying higher catalytic efficiency (kCAT/KM) than the native enzyme complex are identified and selected for production of a C6-C10 alcohol, such as 1-octanol. Combinations of variants of LeuCD' that work together to catalyze the isomerization of all the intermediate 2-alkylmalates (n = 1-5 in intermediate II, FIGURE 1) involved in biosynthesis of an applicable C7-C11 2-ketoacid, such as 2-ketononanoate, are also identified on the basis of in vitro assays.

Example 4


Preparing the modified DC ("DC‴, thiamin dependent decarboxylase) enzyme.



[0058] In this Example 4 embodiment a process similar to that used for the LeuA' enzyme, as described in Example 1, is employed. The process begins with an engineered ketoisovalerate decarboxylase (for example, from Lactococcus lactis subsp. lactis strain IFPL730, GenBank Accession No. AJ746364.1 GI:51870501) or thiamin diphosphate dependent phenylpyruvate decarboxylase (for example, from Saccharomycs cerevisiae (gene YDR380W) or Azospirillum brasilense (GeneBank Accession No. L26240.1). Each engineered variant is made by replacing one or more amino acids within and/or near the active sites by conversion of the original amino acid to glycine, alanine, leucine or valine using site-directed mutagenesis. Variants displaying relatively lower catalytic efficiency (kCAT/KM) for smaller 2-ketoacids (n = 1-5 in intermediate I, FIGURE 1) but which retain or show relatively higher efficiency for a C7-C11 2-ketoacid, such as 2-ketononanoate, are identified and selected for production of a desired C6-C10 alcohol, such as 1-octanol. (It is noted that, in the case of phenylpyruvate decarboxylases, the engineered enzymes may be generally poorer than the wild type enzyme in catalyzing the conversion of phenylpyruvic acid. Each modified enzyme is expressed as a histidine-tagged protein, expressed heterologously in E. coli DE3 cells, and purified using Ni-NTA chromatography. The efficiency of the purified variant enzyme, in decarboxylating a desired C7-C11 2-ketoacids, such as 2-ketononanoate, to form a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted, for example, octanal, is determined by in vitro coupled enzyme assays. One suitable assay method that may be used for this purpose is described in, e.g., Zhang, K.; Sawaya, M.R.; Eisenberg, D.S.; Liao, J.C. "Expanding metabolism for biosynthesis of nonnatural alcohols," Proc. Natl. Acad. Sci. USA, 2008, 105:20653-20658.

Example 5


Preparing the modified ADH ("ADH‴, alcohol dehydrogenase) enzyme.



[0059] This preparation may be accomplished in a manner similar to that used for the LeuA' enzyme, as described in Example 1. In this case the process begins with an engineered human (GenBank Accession No. NP_000662.3 GI:71565154) or E. coli (GenBank Accession No. NC_000913.3 Gene ID: 944988) Class III alcohol dehydrogenase, that is capable of reducing a C6-C10 aldehyde, such as octanal, to a corresponding C6-C10 alcohol, such as 1-octanol. In certain embodiments the starting alcohol dehydrogenase may be an ADH from Saccharomyces cerevisiae. Each engineered variant may then be made by replacing one or more amino acid residues within and/or near the active sites of the selected alcohol dehydrogenase, e.g., a Class III alcohol dehydrogenase, using site-directed mutagenesis. Engineered enzymes will have higher catalytic efficiency (kcat/KM) than the native enzyme in reducing the selected C6-C10 aldehyde, such as octanal, to the corresponding C6-C10 alcohol, such as 1-octanol. Each modified enzyme is then expressed as a histidine-tagged protein, expressed heterologously in E. coli DE3 cells, and purified using Ni-NTA chromatography. The efficiency of the purified variant enzyme in reducing the C6-C10 aldehyde is determined by in vitro enzyme assay. A suitable assay method is described in, e.g., Sanghani, P.C.; Stone, C.L.; Ray, B.D.; Pindel, E.V.; Hurley, T.D.; Bosron, W.F. "Kinetic mechanism of human glutathione-dependent formaldehyde dehydrogenase," Biochemistry, 2000, 39:10720-10729. Variants displaying higher catalytic efficiency (kcat/KM) for the C6-C10 aldehyde are identified and selected for C6-C10 alcohol production.

SEQUENCE LISTING



[0060] 

<110> DOW Global Technologies Inc. Sanghani, Paresh Stowers, Christopher Rodriguez, Brandon Venkateswaran, Amudhan

<120> PROCESSES TO PREPARE ELONGATED 2-KETOACIDS AND C6-C10 COMPOUNDS THEREFROM VIA GENETIC MODIFICATIONS TO MICROBIAL METABOLIC PATHWAYS

<130> 75413-WO-PCT

<150> US 61/915,040
<151> 2013-12-12

<160> 20

<170> PatentIn version 3.5

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Claims

1. A process for preparing a C7-C11 2-ketoacid comprising contacting

a substrate, selected from 2-ketobutyrate and 2-ketoisovalerate, and

(1) a native or genetically modified LeuA enzyme having isopropylmalate synthase activity;

(2) a genetically modified LeuB' enzyme, wherein:

(a) the enzyme is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified

(1) at Leu-96 by substituting glycine;

(2) at Val-198 by substituting alanine;

(3) at Leu-96 by substituting alanine and at Val-198 by substituting alanine;

(4) at Leu-96 by substituting glycine and at Val-198 by substituting alanine;

(5) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or

(6) at Leu-96 by substituting alanine;

or

(b) the enzyme has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (a);
and

(3) a native or genetically modified LeuCD' enzyme complex having isopropylmalate isomerase activity;

under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid.


 
2. The process of claim 1 further comprising contacting the C7-C11 2-ketoacid and a thiamin dependent decarboxylase under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted.
 
3. The process of claim 2 further comprising contacting the C6-C10 aldehyde and at least one enzyme selected from

(1) an alcohol dehydrogenase, under conditions such that the C6-C10 aldehyde is converted to the corresponding C6-C10 alcohol;

(2) an aldehyde dehydrogenase, under conditions such that the C6-C10 aldehyde is converted to the corresponding C6-C10 carboxylic acid;

(3) a fatty aldehyde decarbonylase, under conditions such that the C6-C10 aldehyde is converted to the corresponding C5-C9 alkane; and

(4) combinations thereof.


 
4. A microbial organism that expresses a genetically modified LeuB' enzyme that:

(1) is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified

(i) at Leu-96 by substituting glycine;

(ii) at Val-198 by substituting alanine;

(iii) at Leu-96 by substituting alanine and at Val-198 by substituting alanine;

(iv) at Leu-96 by substituting glycine and at Val-198 by substituting alanine;

(v) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or

(vi) at Leu-96 by substituting alanine;

or

(2) has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, and wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1, the enzyme having been modified as in (1).


 
5. A process to prepare a C6-C10 aldehyde comprising

(1a) contacting a carbon-containing substrate and:

native or genetically modified pyruvate carboxylase having pyruvate carboxylase activity,

native or genetically modified aspartate aminotransferase having aspartate aminotransferase activity,

native or genetically modified bifunctional aspartokinase/homoserine dehydrogenase having bifunctional aspartokinase/homoserine dehydrogenase activity,

native or genetically modified homoserine kinase having homoserine kinase activity,

native or genetically modified threonine synthase having threonine synthase activity,

native or genetically modified aspartate semialdehyde dehydrogenase having aspartate semialdehyde dehydrogenase activity and

native or genetically modified threonine dehydratase having threonine dehydratase activity,

under conditions to form 2-ketobutyrate, or

(1b) contacting a carbon containing substrate and:

native or genetically modified acetolactate synthase isozyme with acetolactate synthase isozyme activity,

ketol-acid reductoisomerase having ketol-acid reductoisomerase activity, and dihydroxy-acid dehydratase having dihydroxy-acid dehydratase activity,

under conditions to form 2-ketoisovalerate;

(2) contacting the 2-ketobutyrate or 2-ketoisovalerate and

(a) a native or genetically modified LeuA enzyme having isopropylmalate synthase activity;

(b) a genetically modified LeuB' enzyme, wherein the LeuB' enzyme

(i) is obtained from Escherichia coli and has an amino acid sequence corresponding to Sequence Listing, SEQ ID 1; the enzyme having been modified

(a) at Leu-96 by substituting glycine;

(b) at Val-198 by substituting alanine;

(c) at Leu-96 by substituting alanine and at Val-198 by substituting alanine;

(d) at Leu-96 by substituting glycine and at Val-198 by substituting alanine;

(e) at Leu-96 by substituting glycine and at Val-198 by substituting glycine; or

(f) at Leu-96 by substituting alanine;

or

(ii) has isopropylmalate dehydrogenase activity and catalyzes the conversion of 3-hexylmalate to form 2-ketononanoate at a catalytic efficiency that is higher than that of a native LeuB enzyme, and wherein the enzyme has an amino acid sequence that is at least 60 percent homologous to the amino acid sequence of Sequence Listing, SEQ ID 1; the enzyme having been modified as in (i); and

(c) a native or genetically modified LeuCD' enzyme complex having isopropylmalate isomerase activity;

under conditions such that the 2-ketobutyrate or 2-ketoisovalerate is converted, via one or more steps, to a C7-C11 2-ketoacid; and

(3) contacting the C7-C11 2-ketoacid and a native or genetically modified thiamin dependent decarboxylase having thiamin dependent decarboxylase activity under conditions such that the C7-C11 2-ketoacid is converted to a C6-C10 aldehyde having one less carbon atom than the C7-C11 2-ketoacid being converted;

each of (1), (2) and (3) occurring in one or more steps;

wherein (1), (2) and (3) independently occur within or outside of a genetically modified microbial organism.


 
6. The process of claim 5 wherein the genetically modified LeuB' enzyme catalyzes the conversion of:

(1) 3-butylmalate to form 2-ketoheptanoate; and/or

(2) 3-pentylmalate to form 2-ketooctanoate;

at a catalytic efficiency that is higher than that of a native LeuB enzyme.
 


Ansprüche

1. Ein Verfahren zum Zubereiten einer C7-C11-2-Ketosäure, beinhaltend das In-Kontakt-Bringen eines Substrats, das aus 2-Ketobutyrat und 2-Ketoisovalerat ausgewählt ist, und von Folgendem:

(1) eines natürlichen oder genetisch veränderten LeuA-Enzyms, das Isopropylmalatsynthaseaktivität aufweist;

(2) eines genetisch veränderten LeuB'-Enzyms, wobei:

(a) das Enzym aus Escherichia coli erhalten wird und eine Aminosäuresequenz aufweist, die Sequenzprotokoll SEQ ID 1 entspricht; wobei das Enzym wie folgt verändert wurde:

(1) an Leu-96 durch Substituieren von Glycin;

(2) an Val-198 durch Substituieren von Alanin;

(3) an Leu-96 durch Substituieren von Alanin und an Val-198 durch Substituieren von Alanin;

(4) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Alanin;

(5) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Glycin; oder

(6) an Leu-96 durch Substituieren von Alanin;

oder

(b) das Enzym Isopropylmalatdehydrogenaseaktivität aufweist und die Umwandlung von 3-Hexylmalat, um 2-Ketononanoat zu bilden, mit einer katalytischen Effizienz katalysiert, die höher als die eines natürlichen LeuB-Enzyms ist, wobei das Enzym eine Aminosäuresequenz aufweist, die mindestens 60 Prozent homolog zu der Aminosäuresequenz von Sequenzprotokoll SEQ ID 1 ist; wobei das Enzym wie in (a) verändert wurde;
und

(3) eines natürlichen oder genetisch veränderten LeuCD'-Enzymkomplexes, der Isopropylmalatisomeraseaktivität aufweist;

unter solchen Bedingungen, dass das 2-Ketobutyrat oder 2-Ketoisovalerat über einen oder mehrere Schritte in eine C7-C11-2-Ketosäure umgewandelt wird.
 
2. Verfahren gemäß Anspruch 1, das ferner das In-Kontakt-Bringen der C7-C11-2-Ketosäure und einer thiaminabhängigen Decarboxylase unter solchen Bedingungen beinhaltet, dass die C7-C11-2-Ketosäure in ein C6-C10-Aldehyd umgewandelt wird, das ein Kohlenstoffatom weniger aufweist als die in der Umwandlung befindliche C7-C11-2-Ketosäure.
 
3. Verfahren gemäß Anspruch 2, das ferner das In-Kontakt-Bringen des C6-C10-Aldehyds und mindestens eines Enzyms beinhaltet, das aus Folgendem ausgewählt ist:

(1) einer Alkoholdehydrogenase, unter solchen Bedingungen, dass das C6-C10-Aldehyd in den entsprechenden C6-C10-Alkohol umgewandelt wird;

(2) einer Aldehyddehydrogenase, unter solchen Bedingungen, dass das C6-C10-Aldehyd in die entsprechende C6-C10-Carbonsäure umgewandelt wird;

(3) einer Fettaldehyddecarbonylase, unter solchen Bedingungen, dass das C6-C10-Aldehyd in das entsprechende C5-C9-Alkan umgewandelt wird; und

(4) Kombinationen davon.


 
4. Ein Mikrobenorganismus, der ein genetisch verändertes LeuB'-Enzym ausdrückt, das:

(1) aus Escherichia coli erhalten wird und eine Aminosäuresequenz aufweist, die Sequenzprotokoll SEQ ID 1 entspricht; wobei das Enzym wie folgt verändert wurde:

(i) an Leu-96 durch Substituieren von Glycin;

(ii) an Val-198 durch Substituieren von Alanin;

(iii) an Leu-96 durch Substituieren von Alanin und an Val-198 durch Substituieren von Alanin;

(iv) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Alanin;

(v) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Glycin; oder

(vi) an Leu-96 durch Substituieren von Alanin;

oder

(2) Isopropylmalatdehydrogenaseaktivität aufweist und die Umwandlung von 3-Hexylmalat, um 2-Ketononanoat zu bilden, mit einer katalytischen Effizienz katalysiert, die höher als die eines natürlichen LeuB-Enzyms ist, und wobei das Enzym eine Aminosäuresequenz aufweist, die mindestens 60 Prozent homolog zu der Aminosäuresequenz von Sequenzprotokoll SEQ ID 1 ist, wobei das Enzym wie in (1) verändert wurde.


 
5. Ein Verfahren zum Zubereiten eines C6-C10-Aldehyds, das Folgendes beinhaltet:

(1a) In-Kontakt-Bringen eines kohlenstoffhaltigen Substrats und:

natürlicher oder genetisch veränderter Pyruvatcarboxylase, die Pyruvatcarboxylaseaktivität aufweist,

natürlicher oder genetisch veränderter Aspartataminotransferase, die Aspartataminotransferaseaktivität aufweist,

natürlicher oder genetisch veränderter bifunktioneller Aspartokinase-Homoserin-Dehydrogenase, die bifunktionelle Aspartokinase-Homoserin-Dehydrogenaseaktivität aufweist,

natürlicher oder genetisch veränderter Homoserinkinase, die Homoserinkinaseaktivität aufweist,

natürlicher oder genetisch veränderter Threoninsynthase, die Threoninsynthaseaktivität aufweist,

natürlicher oder genetisch veränderter Aspartatsemialdehyddehydrogenase, die Aspartatsemialdehyddehydrogenaseaktivität aufweist, und

natürlicher oder genetisch veränderter Threonindehydratase, die Threonindehydrataseaktivität aufweist,

unter Bedingungen, um 2-Ketobutyrat zu bilden, oder

(1b) In-Kontakt-Bringen eines kohlenstoffhaltigen Substrats und:

eines natürlichen oder genetisch veränderten Acetolactatsynthaseisozyms mit Acetolactatsynthaseisozymaktivität,

Ketolsäurereductoisomerase, die Ketolsäurereductoisomeraseaktivität aufweist, und

Dihydroxysäuredehydratase, die Dihydroxysäuredehydrataseaktivität aufweist,

unter Bedingungen, um 2-Ketoisovalerat zu bilden;

(2) In-Kontakt-Bringen des 2-Ketobutyrats oder 2-Ketoisovalerats und:

(a) eines natürlichen oder genetisch veränderten LeuA-Enzyms, das Isopropylmalatsynthaseaktivität aufweist;

(b) eines genetisch veränderten LeuB'-Enzyms, wobei das LeuB'-Enzym:

(i) aus Escherichia coli erhalten wird und eine Aminosäuresequenz aufweist, die Sequenzprotokoll SEQ ID 1 entspricht; wobei das Enzym wie folgt verändert wurde:

(a) an Leu-96 durch Substituieren von Glycin;

(b) an Val-198 durch Substituieren von Alanin;

(c) an Leu-96 durch Substituieren von Alanin und an Val-198 durch Substituieren von Alanin;

(d) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Alanin;

(e) an Leu-96 durch Substituieren von Glycin und an Val-198 durch Substituieren von Glycin; oder

(f) an Leu-96 durch Substituieren von Alanin;

oder

(ii) Isopropylmalatdehydrogenaseaktivität aufweist und die Umwandlung von 3-Hexylmalat, um 2-Ketononanoat zu bilden, mit einer katalytischen Effizienz katalysiert, die höher als die eines natürlichen LeuB-Enzyms ist, und wobei das Enzym eine Aminosäuresequenz aufweist, die mindestens 60 Prozent homolog zu der Aminosäuresequenz von Sequenzprotokoll SEQ ID 1 ist; wobei das Enzym wie in (i) verändert wurde; und

(c) eines natürlichen oder genetisch veränderten LeuCD'-Enzymkomplexes, der Isopropylmalatisomeraseaktivität aufweist;

unter solchen Bedingungen, dass das 2-Ketobutyrat oder 2-Ketoisovalerat über einen oder mehrere Schritte in eine C7-C11-2-Ketosäure umgewandelt wird; und

(3) In-Kontakt-Bringen der C7-C11-2-Ketosäure und einer natürlichen oder genetisch veränderten thiaminabhängigen Decarboxylase, die thiaminabhängige Decarboxylaseaktivität aufweist, unter solchen Bedingungen, dass die C7-C11-2-Ketosäure in ein C6-C10-Aldehyd umgewandelt wird, das ein Kohlenstoffatom weniger aufweist als die in der Umwandlung befindliche C7-C11-2-Ketosäure;

wobei jedes von (1), (2) und (3) in einem oder mehreren Schritten erfolgt;

wobei (1), (2) und (3) unabhängig innerhalb oder außerhalb eines genetisch veränderten Mikrobenorganismus erfolgen.


 
6. Verfahren gemäß Anspruch 5, wobei das genetisch veränderte LeuB'-Enzym die Umwandlung von Folgendem:

(1) 3-Butylmalat, um 2-Ketoheptanoat zu bilden; und/oder

(2) 3-Pentylmalat, um 2-Ketooctanoat zu bilden;

mit einer katalytischen Effizienz katalysiert, die höher ist als die eines natürlichen LeuB-Enzyms.
 


Revendications

1. Un procédé pour préparer un 2-cétoacide en C7-C11 comprenant le fait de mettre en contact un substrat, sélectionné parmi le 2-cétobutyrate et le 2-cétoisovalérate, et

(1) une enzyme LeuA native ou génétiquement modifiée ayant une activité isopropylmalate synthase ;

(2) une enzyme LeuB' génétiquement modifiée, où :

(a) l'enzyme est obtenue à partir d'Escherichia coli et a une séquence d'acides aminés correspondant à la liste de séquences, SEQ ID 1 ; l'enzyme ayant été modifiée

(1) au niveau de Leu-96 par substitution de glycine ;

(2) au niveau de Val-198 par substitution d'alanine ;

(3) au niveau de Leu-96 par substitution d'alanine et au niveau de Val-198 par substitution d'alanine ;

(4) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution d'alanine ;

(5) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution de glycine ; ou

(6) au niveau de Leu-96 par substitution d'alanine ;

ou

(b) l'enzyme a une activité isopropylmalate déshydrogénase et catalyse la conversion de 3-hexylmalate afin de former le 2-cétononanoate à une efficacité catalytique qui est plus élevée que celle d'une enzyme LeuB native, où l'enzyme a une séquence d'acides aminés qui est au moins de 60 pour cent homologue à la séquence d'acides aminés de la liste de séquences, SEQ ID 1 ; l'enzyme ayant été modifiée tel que dans (a) ;
et

(3) un complexe d'enzyme LeuCD' native ou génétiquement modifiée ayant une activité isopropylmalate isomérase ;

dans des conditions telles que le 2-cétobutyrate ou le 2-cétoisovalérate soit converti, par le biais d'une ou de plusieurs étapes, en un 2-cétoacide en C7-C11.
 
2. Le procédé de la revendication 1 comprenant en outre le fait de mettre en contact le 2-cétoacide en C7-C11 et une décarboxylase thiamine-dépendante dans des conditions telles que le 2-cétoacide en C7-C11 soit converti en un aldéhyde en C6-C10 ayant un atome de carbone de moins que le 2-cétoacide en C7-C11 étant converti.
 
3. Le procédé de la revendication 2 comprenant en outre le fait de mettre en contact l'aldéhyde en C6-C10 et au moins une enzyme sélectionnée parmi

(1) une alcool déshydrogénase, dans des conditions telles que l'aldéhyde en C6-C10 soit converti en l'alcool en C6-C10 correspondant ;

(2) une aldéhyde déshydrogénase, dans des conditions telles que l'aldéhyde en C6-C10 soit converti en l'acide carboxylique en C6-C10 correspondant ;

(3) une aldéhyde gras décarbonylase, dans des conditions telles que l'aldéhyde en C6-C10 soit converti en l'alcane en C5-C9 correspondant ; et

(4) des combinaisons de celles-ci.


 
4. Un organisme microbien qui exprime une enzyme LeuB' génétiquement modifiée qui :

(1) est obtenue à partir d'Escherichia coli et a une séquence d'acides aminés correspondant à la liste de séquences, SEQ ID 1 ; l'enzyme ayant été modifiée

(i) au niveau de Leu-96 par substitution de glycine ;

(ii) au niveau de Val-198 par substitution d'alanine ;

(iii) au niveau de Leu-96 par substitution d'alanine et au niveau de Val-198 par substitution d'alanine ;

(iv) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution d'alanine ;

(v) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution de glycine ; ou

(vi) au niveau de Leu-96 par substitution d'alanine ;

ou

(2) a une activité isopropylmalate déshydrogénase et catalyse la conversion de 3-hexylmalate afin de former le 2-cétononanoate à une efficacité catalytique qui est plus élevée que celle d'une enzyme LeuB native, et où l'enzyme a une séquence d'acides aminés qui est au moins de 60 pour cent homologue à la séquence d'acides aminés de la liste de séquences, SEQ ID 1, l'enzyme ayant été modifiée tel que dans (1).


 
5. Un procédé pour préparer un aldéhyde en C6-C10 comprenant

(1a) le fait de mettre en contact un substrat contenant du carbone et :

une pyruvate carboxylase native ou génétiquement modifiée ayant une activité pyruvate carboxylase,

une aspartate aminotransférase native ou génétiquement modifiée ayant une activité aspartate aminotransférase,

une aspartokinase/homosérine déshydrogénase bifonctionnelle native ou

génétiquement modifiée ayant une activité aspartokinase/homosérine déshydrogénase bifonctionnelle,

une homosérine kinase native ou génétiquement modifiée ayant une activité homosérine kinase,

une thréonine synthase native ou génétiquement modifiée ayant une activité thréonine synthase,

une aspartate semialdéhyde déshydrogénase native ou génétiquement modifiée ayant une activité aspartate semialdéhyde déshydrogénase et

une thréonine déshydratase native ou génétiquement modifiée ayant une activité thréonine déshydratase,

dans des conditions afin de former le 2-cétobutyrate, ou

(1b) le fait de mettre en contact un substrat contenant du carbone et :

une isozyme acétolactate synthase native ou génétiquement modifiée avec une activité isozyme acétolactate synthase,

une cétol-acide réductoisomérase ayant une activité cétol-acide réductoisomérase, et

une dihydroxy-acide déshydratase ayant une activité dihydroxy-acide déshydratase,

dans des conditions afin de former le 2-cétoisovalérate ;

(2) le fait de mettre en contact le 2-cétobutyrate ou le 2-cétoisovalérate et

(a) une enzyme LeuA native ou génétiquement modifiée ayant une activité isopropylmalate synthase ;

(b) une enzyme LeuB' génétiquement modifiée, où l'enzyme LeuB'

(i) est obtenue à partir d'Escherichia coli et a une séquence d'acides aminés correspondant à la liste de séquences, SEQ ID 1 ; l'enzyme ayant été modifiée

(a) au niveau de Leu-96 par substitution de glycine ;

(b) au niveau de Val-198 par substitution d'alanine ;

(c) au niveau de Leu-96 par substitution d'alanine et au niveau de Val-198 par substitution d'alanine ;

(d) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution d'alanine ;

(e) au niveau de Leu-96 par substitution de glycine et au niveau de Val-198 par substitution de glycine ; ou

(f) au niveau de Leu-96 par substitution d'alanine ;

ou

(ii) a une activité isopropylmalate déshydrogénase et catalyse la conversion de 3-hexylmalate afin de former le 2-cétononanoate à une efficacité catalytique qui est plus élevée que celle d'une enzyme LeuB native, et où l'enzyme a une séquence d'acides aminés qui est au moins de 60 pour cent homologue à la séquence d'acides aminés de la liste de séquences, SEQ ID 1 ; l'enzyme ayant été modifiée tel que dans (i) ; et

(c) un complexe d'enzyme LeuCD' native ou génétiquement modifiée ayant une activité isopropylmalate isomérase ;

dans des conditions telles que le 2-cétobutyrate ou le 2-cétoisovalérate soit converti, par le biais d'une ou de plusieurs étapes, en un 2-cétoacide en C7-C11 ; et

(3) le fait de mettre en contact le 2-cétoacide en C7-C11 et une décarboxylase thiamine-dépendante native ou génétiquement modifiée ayant une activité décarboxylase thiamine-dépendante dans des conditions telles que le 2-cétoacide en C7-C11 soit converti en un aldéhyde en C6-C10 ayant un atome de carbone de moins que le 2-cétoacide en C7-C11 étant converti ;

chaque action parmi (1), (2) et (3) se produisant en une ou plusieurs étapes ;

où (1), (2) et (3) se produisent indépendamment au sein ou à l'extérieur d'un organisme microbien génétiquement modifié.


 
6. Le procédé de la revendication 5 où l'enzyme LeuB' génétiquement modifiée catalyse la conversion de :

(1) 3-butylmalate afin de former le 2-cétoheptanoate ; et/ou

(2) 3-pentylmalate afin de former le 2-cétooctanoate ;

à une efficacité catalytique qui est plus élevée que celle d'une enzyme LeuB native.
 




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