GENETICALLY MODIFIED PHENYLPYRUVATE DECARBOXYLASE, PROCESSES TO PREPARE, AND USES THEREOF

Abstract

Modification of the amino acid sequence of a phenylpyruvate decarboxylase from Azospirillum brasilense produces a novel group of phenylpyruvate decarboxylases with improved specificity to certain substrates, including in particular C7-C11 2-ketoacids such as, for example, 2-ketononanoate and 2-keto-octanoate. This specificity enables effective use of the phenylpyruvate decarboxylase in, for example, an in vivo process wherein 2-ketobutyrate or 2-ketoisovalerate are converted to C7-C11 2-ketoacids, and the novel phenylpyruvate decarboxylase converts the C7-C11 2-ketoacid to a C6-C10 aldehyde having one less carbon than the 2-ketoacid. Ultimately, through contact with additional enzymes, such C6-C10 aldehydes may be converted to, for example, C6-C10 alcohols, C6-C10 carboxylic acids, C6-C10 alkanes, and other derivatives. Use of the novel genetically modified phenylpyruvate de carboxylases may represent a lower cost alternative to non-biobased approaches.

Claims

1. A process for genetically modifying a microorganism comprising: (A) selecting a microorganism that produces a C.sub.7-C.sub.11 2-ketoacid; and (B) inserting a non-native nucleic acid sequence that encodes at least one of: i. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F385L, and having phenylpyruvate decarboxylase activity; ii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461C, and having phenylpyruvate decarboxylase activity; iii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461V, and having phenylpyruvate decarboxylase activity; iv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461L, and having phenylpyruvate decarboxylase activity; v. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532V, and having phenylpyruvate decarboxylase activity; vi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532L, and having phenylpyruvate decarboxylase activity; vii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536G, and having phenylpyruvate decarboxylase activity; viii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536A, and having phenylpyruvate decarboxylase activity; ix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536L, and having phenylpyruvate decarboxylase activity; x. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536I, and having phenylpyruvate decarboxylase activity; xi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536V, and having phenylpyruvate decarboxylase activity; xii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532V and Q536V, and having phenylpyruvate decarboxylase activity; xiii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M380V and M461V, and having phenylpyruvate decarboxylase activity; xiv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and M461V, and having phenylpyruvate decarboxylase activity; xv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V, and having phenylpyruvate decarboxylase activity; xvi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V, and having phenylpyruvate decarboxylase activity; xvii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and Q536V, and having phenylpyruvate decarboxylase activity; xviii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461L and Q536V, and having phenylpyruvate decarboxylase activity; xix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461A and Q536V, and having phenylpyruvate decarboxylase activity; xx. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V and F532V, and having phenylpyruvate decarboxylase activity; xxi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F465L and Q536V, and having phenylpyruvate decarboxylase activity; xxii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V, F532V, and Q536V, and having phenylpyruvate decarboxylase activity; such that a non-native phenylpyruvate decarboxylase is expressed in the microorganism.

2. The process of claim 1 wherein: (A) the microorganism is Escherichia coli; (B) the non-native nucleic acid sequence is obtained from Azospirillum brasilense; and (C) the non-native phenylpyruvate decarboxylase takes part in a metabolic pathway that converts the C.sub.7-C.sub.11 2-ketoacids to C.sub.6-C.sub.10 aldehydes having one less carbon atom than the C.sub.7-C.sub.11 2-ketoacid being converted.

3. The process of claim 2, wherein the metabolic pathway proceeds during anaerobic fermentation.

4. A genetically modified microorganisms produced by the process of claim 1.

5. (canceled)

6. The process of claim 1 wherein (A) the microrganism is a Clostridium species; (B) the non-native phenylpyruvate decarboxylase takes part in metabolic pathway that includes a Wood-Ljungdahl pathway; and (C) the non-native nucleic acid sequence is obtained from Azospirillum basilense.

7. A process to prepare a C.sub.6-C.sub.10 aldehyde comprising the steps of: (A) contacting a C.sub.4-C.sub.10 2-ketoacid substrate, an isopropylmalate synthase, an isopropylmalate isomerase, and an isopropylmalate dehydrogenase, under conditions that the C.sub.4-C.sub.10 2-ketoacid substrate is converted to a C.sub.7-C.sub.11 2-ketoacid through one or more biochemical reactions; (B) contacting the C.sub.7-C.sub.11 2-ketoacid and a phenlypyruvate decarboxylase, the phenylpyruvate decarboxylase comprising at least one of: i. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F385L, and having phenylpyruvate decarboxylase activity; ii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461C, and having phenylpyruvate decarboxylase activity; iii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461V, and having phenylpyruvate decarboxylase activity; iv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461L, and having phenylpyruvate decarboxylase activity; v. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532V, and having phenylpyruvate decarboxylase activity; vi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532L, and having phenylpyruvate decarboxylase activity; vii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536G, and having phenylpyruvate decarboxylase activity; viii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536A, and having phenylpyruvate decarboxylase activity; ix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536L, and having phenylpyruvate decarboxylase activity; x. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536I, and having phenylpyruvate decarboxylase activity; xi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536V, and having phenylpyruvate decarboxylase activity; xii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532V and Q536V, and having phenylpyruvate decarboxylase activity; xiii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M380V and M461V, and having phenylpyruvate decarboxylase activity; xiv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and M461V, and having phenylpyruvate decarboxylase activity; xv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V, and having phenylpyruvate decarboxylase activity; xvi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V, and having phenylpyruvate decarboxylase activity; xvii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and Q536V, and having phenylpyruvate decarboxylase activity; xviii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461L and Q536V, and having phenylpyruvate decarboxylase activity; xix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461A and Q536V, and having phenylpyruvate decarboxylase activity; xx. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V and F532V, and having phenylpyruvate decarboxylase activity; xxi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F465L and Q536V, and having phenylpyruvate decarboxylase activity; or xxii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V, F532V, and Q536V, and having phenylpyruvate decarboxylase activity; under conditions such that the C.sub.7-C.sub.11 2-ketoacid is converted to a C.sub.6-C.sub.10 aldehyde having one less carbon atom than the C.sub.7-C.sub.11 2-ketoacid being converted.

8. (canceled)

9. A genetically modified phenylpyruvate decarboxylase polypeptide having phenylpyruvate decarboxylase activity, the polypeptide comprising at least one of: i. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F385L; ii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461C; iii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461V; iv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation M461L; v. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532V; vi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation F532L; vii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536G; viii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536A; ix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536L; x. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536I; xi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutation Q536V; xii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532V and Q536V; xiii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M380V and M461V; xiv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and M461V; xv. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V; xvi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F532A and Q536V; xvii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F385L and Q536V; xviii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461L and Q536V; xix. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461A and Q536V; xx. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V and F532V; xxi. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations F465L and Q536V; or xxii. an amino acid sequence comprising at least 90 percent homology to SEQ ID 4 and comprising the mutations M461V, F532V, and Q536V.

10. The polypeptide of claim 9 wherein the amino acid sequence is at least 95 percent homologous thereto.

11. The process according of claim 7, wherein (A) and (B) independently occur within or outside of a genetically modified microbial organism.

12. The process of claim 7, wherein the at least one of the C.sub.4-C.sub.10 2-ketoacid substrate comprises 2-ketobutyrate.

13. The process of claim 7, wherein the at least one of the C.sub.4-C.sub.10 2-ketoacid substrate comprises 2-ketoisovalerate.

14. The process according to claim 7, further comprising: (III) providing the C.sub.6-C.sub.10 aldehyde with an alcohol dehydrogenase having alcohol dehydrogenase activity, under conditions that the C.sub.6-C.sub.10 aldehyde is converted to a corresponding C.sub.6-C.sub.10 alcohol.

15. The process according to claim 7, further comprising: (III) providing the C.sub.6-C.sub.10 aldehyde with an aldehyde dehydrogenase having aldehyde dehydrogenase activity, under conditions that the C.sub.6-C.sub.10 aldehyde is converted to a corresponding C.sub.6-C.sub.10 carboxylic acid.

16. The process according to claim 7, further comprising: (III) providing the C.sub.6-C.sub.10 aldehyde with an aldehyde decarbonylase having fatty aldehyde decarbonylase activity, under conditions that the C.sub.6-C.sub.10 aldehyde is converted to a corresponding C.sub.n-1 alkane.

17. The process according to claim 7, wherein the process occurs under aerobic or anaerobic conditions.

18. A genetically modified microorganism produced by the process of claim 2.

19. A genetically modified microorganism produced by the process of claim 6.

Description

[0064] FIG. 1 illustrates chain elongation by recursive (iterative) activity followed by decarboxylation to an aldehyde that is one carbon atom shorter than the 2-ketoacid preceding it in the iterative pathway.

[0065] FIG. 2 illustrates linear C5-C8 alcohol production from 2-ketobutyrate in vitro using a combination of isopropylmalate synthase, isopropylmalate isomerase, isopropylmalate dehydrogenase, and alcohol dehydrogenase (ADH6) in combination with the F385L variant of AbPPDC (SEQ ID 8).

[0066] FIG. 3 illustrates branched C5-C8 alcohol production from 2-ketoisovalerate in vitro using a combination of isopropylmalate synthase, isopropylmalate isomerase, isopropylmalate dehydrogenase, and alcohol dehydrogenase (ADH6) in combination with the F385L variant of AbPPDC (SEQ ID 8).

[0067] FIG. 4 illustrates branched C5-C8 alcohol production from 3-methyl-2-ketopentanoate in vitro using a combination of isopropylmalate synthase, isopropylmalate isomerase, isopropylmalate dehydrogenase, and alcohol dehydrogenase (ADH6) in combination with the F385L variant of AbPPDC (SEQ ID 8).

[0068] FIG. 5 illustrates the mean alcohol distributions for serum bottle fermentations of E. coli containing the “+1 pathway” enzymes in combination with AbPPDC wild type (WT), AbPPDC variants, KIVD WT (Lactococcus lactis keto-isovalerate decarboxylase), and no decarboxylase. ADH6 is also included in all strain constructs.

[0069] In general the present invention includes, among other things, two specific embodiments of a novel phenylpyruvate decarboxylase that may be, in the first embodiment, the expression of an amino acid sequence that is obtained from Azospirillum brasilense (A. brasilense), corresponding to GenBank: Accession No. L26240, or is at least 80 percent (%) homologous thereto. In a second embodiment, the present invention includes the previously defined genetically modified phenylpyruvate decarboxylase, but with further intentional genetic engineering to insert one, two or three modifications of specific amino acids within the sequence, which again serve to modify the catalytic efficiency of the phenylpyruvate decarboxylase in ways that are in many cases advantageous in carrying out a variety of biosyntheses wherein the phenylpyruvate decarboxylase participates. In particular, either the wild type or the nucleic acid-modified Azospirillum brasilense phenylpyruvate decarboxylase enzymes can be used in combination with isopropylmalate synthase, isopropylmalate isomerase and isopropylmalate dehydrogenase enzymes to produce alcohols, carboxylic acids or alkanes.

[0070] As will be shown herein, novel phenylpyruvate decarboxylase enzymes with improved properties over the wild type enzyme of a selected host microorganism were created through genetic modification in one of a variety of ways that are described herein; or is an enzyme represented by an amino acid sequence that is at least 80% homologous to the A. brasilense phenylpyruvate decarboxylase and includes the same modifications; processes to make it via recombinant, engineered, or technology combining both recombinant and engineered approaches; processes to make C6-C10 alcohols, carboxylic acids and alkanes using the wild type or novel phenylpyruvate decarboxylase; and a genetically modified microbial organism that can express or overexpress this enzyme and can be used to produce C6-C10 alcohols, carboxylic acids and alkanes. As the term is used herein, homology refers to identical or functional correspondence of 80 percent, or more, of the amino acids listed in the sequence, in their given positions.

[0071] The novel phenylpyruvate decarboxylase may be used or expressed as part of, in certain particular embodiments, a metabolic pathway that produces acetyl co-A via either an anabolic (e.g., Wood-Ljundahl) or catabolic (e.g., glycolysis, or a pentose phosphate pathway) route, and ultimately takes part in conversion of a C7-C11 2-ketoacid to form the corresponding C6-10 aldehyde having one less carbon. In some embodiments the C6-C10 aldehyde may be further reacted to form a C6-C10 alcohol, carboxylic acid or alkane. Because of the specific alterations in its amino acid sequence that are described herein, the genetically modified phenylpyruvate decarboxylases of the invention offer some significant differences in specificity to various substrates, and this alteration in specificity offers important advantages in terms of product yields and the reduction or elimination of undesirable and/or competing side products.

[0072] The invention includes a number of altered amino acid sequences of A. brasilense phenylpyruvate decarboxylase that have been identified as exhibiting improved decarboxylations of C7-C11 2-ketoacids in comparison with the wild type A. brasilense amino acid sequence corresponding to GenBank: Accession No. L26240, which is shown in SEQ ID 4. Six sites within the wild type sequence have been identified as key to obtaining the improvements. These are: Met-380, Phe-385, Met-461, Phe-465, Phe-532, Gln-536, and combinations thereof. In each alteration changes are made wherein either valine, leucine, alanine, glycine, or isoleucine are substituted at the identified site(s) for the wild type amino acid, with substitutions varying from single-site (i.e., single amino acid constituting three base pairs) substitution, to a wide variety of multiple-site (from 2 to 5 sites) substitutions defined as “combinations” of the identified sites, preferably from 2 to 3. SEQ ID 3-82 show amino acid sequences for the many variations produced that include one or more of the substitutions as specified. The substitutions can be summarized as follows: (1) substituting Met-380 with valine; or (2) substituting Phe-385 with valine, leucine or isoleucine; or (3) substituting Met-461 with valine, leucine, alanine, or cysteine; or (4) substituting Phe-465 with valine or leucine; or (5) substituting Phe-532 with glycine, alanine, valine, or leucine; or (6) substituting Gln-536 with valine, leucine, isoleucine, alanine, or glycine; or (7) any combination of three substitutions as described in (1)-(6);

[0073] It will be understood by those skilled in the art that the inventive genetically modified phenylpyruvate decarboxylases may be used either in vivo, i.e., by a genetically modified microorganism, or in vitro. In view of this, the terms “genetically modified,” or “modified,” as used herein, refer to the group of inventive phenylpyruvate decarboxylases having an intentionally altered amino acid sequence, i.e., a “non-wild type” amino acid sequence, or a microbial organism (depending upon placement of either term as an adjective) having a genome that has been intentionally altered as to (at least) the specific, modified decarboxylase(s) described and defined as inventive herein. Such alteration may have been accomplished via recombinant technology, where one or more genes is transferred from a second, different microbial organism into a target microbial organism; or engineered technology, wherein the nucleic acids within the target microbial organism are altered, generally via site-directed mutagenesis, resulting in the conversion of at least one nucleic acid to a different nucleic acid and therefore modification of one or more enzymes. With today's DNA synthesis technologies, recombinant technology can also be accomplished using fully synthetic DNA that is transferred to the target microorganism using conventional methods. Combinations of any of the above methods may also be employed.

[0074] The invention further includes a process to prepare C6-C10 aldehydes, C6-C10 carboxylic acids, C6-C10 alkanes, and C6-10 alcohols such as hexanol, heptanol and/or 1-octanol, via contact between a starting substrate and a series of enzymes that include one or more of the genetically-modified phenylpyruvate decarboxylases of the invention to ultimately convert that substrate, using additional enzymes and steps, to the desired C6-C10 aldehyde, alcohol, carboxylic acid, or alkane. This 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 production of the C6-C10 alcohol(s), carboxylic acid(s), or alkane(s), may be carried out via in vitro methodology, typically beginning from a starting point that does not include a microbial organism.

[0075] In order to obtain the group of modified phenylpyruvate decarboxylases of the invention, it is desirable, in one embodiment, to perform a protocol similar to that described hereunder. In general the examples show genetic modification involving engineering to alter one or more nucleic acid base(s) in a given codon in order to alter the enzyme of which the nucleic acid base(s) is/are a part. Such may be used simply to produce altered enzyme for, e.g., in vitro assay purposes. In contrast, the genome of a host microbial organism may be preferably altered for a larger scale production strain.

[0076] The following methodology, designed for in vitro enzyme production, may be carried out as is generally understood by those skilled in the art. In general, a suitable database, such as GenBank, is used to obtain the genetic codes for the wild type 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 wherein the alteration(s) of the nucleic acid base(s) is/are done via site-directed mutagenesis. The variant-type enzymes must then be subjected to purification to separate out non-targeted proteins, leaving a purified enzyme that will exhibit a higher-than-wild type catalytic efficiency. This can be appropriately assayed in vitro, according to the methodology most suitable for 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 conversion of a C7-C11 2-ketoacid to the corresponding C6-C10 aldehyde having one less carbon (e.g., converting 2-ketononanoate to octanal, or 2-ketooctanoate to heptanal) which can then be reduced, in one embodiment, by contact with an appropriate wild type or non-wild type alcohol dehydrogenase, to form the corresponding C6-C10 alcohol.

[0077] 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, although in some cases an in vitro approach may be suitable for commercial scale production. Frequently, an in vitro approach may be particularly convenient for laboratory and general research purposes, such as to carry out enzymatic assays. For example, desirable microbial organism, useful for large or commercial scale fermentative production of an enzyme-facilitated product, such as, in certain particular embodiments, a C6-C10 alcohol or combination of C6-C10 alcohols, may be prepared. Such preparation may be carried out by inserting the DNA, or pieces of DNA, which encode for the desired improved enzyme, from a first microbial organism into the genome of a second, “host” microbial organism known or believed to possess one or more desired metabolic pathways and/other desired features, such as inhibition-resistant fermentative capability, using recombinant technology. In general the in vivo approach employs such a microbial organism's wild type 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.

[0078] For example, in one embodiment a suitable carbon-containing substrate, such as a C5 or C6 sugar (e.g., glucose, sucrose, pentose, or a combination thereof), may be converted directly to pyruvate via one of the catabolic or anabolic pathways, such as a glycolysis or pentose phosphate pathway. 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; ThrC, which is threonine synthase; and ASD, which is 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-keto-isovalerate via the activities of IlvBN/IlvGM, IlvC, and IlvD in leucine biosynthesis. See, also, Zhang, K.; Sawaya, M. R.; et al., ibid.

[0079] From this point a wild type or genetically modified form of one or more of the three enzymes within the leucine biosynthetic pathway, that are involved in elongating 2-ketoacids, operate to convert the 2-ketobutyrate or 2-ketoisovalerate to a C7-C11 2-ketoacid. These enzymes are generally referred to, without reference to any specific microbial organism, as isopropylmalate synthase, isopropylmalate isomerase, and isopropylmalate dehydrogenase. However, in E. coli specifically, they are referred to as LeuA (GenBank:Accession No. NC 000913.3 Gene ID: 947465), LeuB (GenBank:Accession No. NC 000913.3 Gene ID: 944798), and LeuCD (GenBank:Accession No. NC 000913.3 Gene ID: 945076 and Gene ID: 945642), respectively. One example of this chain elongation is shown in FIG. 1, wherein 2-ketobutyrate is converted, via a number of steps termed a “+1 pathway,” to the C7-C11 2-ketoacid 2-ketononanoate.

[0080] In certain particular embodiments the wild type enzymes of leucine biosynthetic pathway involved in extending 2-ketoacids may be modified, in particular by inclusion of at least one exogenous enzyme, enzyme complex, or combination thereof, to convert 2-ketobutyrate first to 2-ketovalerate, then to 2-ketocaproate, then to 2-ketoheptanoate and continuing, if desired, to another elongated 2-ketoacid up to 2-ketoundecanoate, i.e., a desired C7-C11 2-ketoacid, as chain-lengthening occurs. However, it is optionally possible to modify only one or two of the enzymes, enzyme complex, or combination thereof, in order to obtain acceptable or desirable production of the C7-C11 2-ketoacid. These enzymes may include LeuA, LeuB and/or LeuCD, as mentioned hereinabove.

[0081] Particularly applicable to modification of this portion of the pathway is the disclosure of co-pending International Patent Application Serial No. PCT/US14/69438, filed Dec. 10, 2014 (Attorney Docket No. 75413-WO-PCT), claiming the benefit of U.S. Provisional Patent Application No. 61/915,040, filed Dec. 12, 2013 (Attorney Docket No. 75413-US-PSP), which are both incorporated herein in their entireties by reference. In certain embodiments, at least a modified isopropylmalate dehydrogenase variant (which is the product of the LeuB gene in E. coli) is selected, or in other embodiments at least a modified LeuA (LeuA′) and LeuB′ variant is included, preferably, but not necessarily, as described in one or both of the referenced patent applications. It is also preferable to employ other combinations of the LeuA′, LeuB′ and modified LeuCD (LeuCD′) enzymes/enzyme complex. Again, it should be noted that the “Leu”+letter (A, B, CD) designations are specific names for the leucine pathway enzymes of isopropylmalate synthase, isopropylmalate isomerase, and isopropylmalate dehydrogenase in E. coli, while the same or equivalent enzymes in the leucine pathway of other organisms may have different names.

[0082] Finally, the inventive genetically modified phenylpyruvate decarboxylase may, in this particular embodiment, serve to convert the C7-C11 2-ketoacid to an aldehyde having one less carbon than the substrate 2-ketoacid. In various embodiments, the resulting C6-C10 aldehyde may find a wide variety of uses, as a product in itself or as a starting or intermediate product for the production of products including the C6-C10 alcohols. Preparation of C6-C10 alcohols may be accomplished via conversion of the C6-C10 aldehyde by an appropriate wild type or genetically modified alcohol dehydrogenase, but other products, such as C6-C10 alkanes, may also be prepared, via the action or expression of a fatty aldehyde decarbonylase, or C6-10 carboxylic acids may be prepared by the action or expression of an aldehyde dehydrogenase. See, e.g., Choi, Y. J.; Lee, S. Y. “Microbial production of short-chain alkanes,” Nature, 2013, 502:571-574. Thus, the C6-10 aldehydes are industrially highly useful as excellent intermediate products for preparing a wide variety of other products.

[0083] Accordingly, it is anticipated that the inventive family of genetically modified phenylpyruvate decarboxylases will be applicable in a wide variety of industries. Such industries may include, for example, use in fuels, plastics, food, packaging, cosmetics, perfumes, pharmaceuticals, cleaning materials, pollution control, perfumes, drugs, and many others. While there are a number of possible amino acid sequences falling fully within the scope of the claims of the present invention, it is noted that certain amino acid sequences, identified by their sequence identification numbers (SEQ ID) as selected from SEQ ID 34, 36, 38, 40, 42, 46, 62, 68, and 76, are particularly well-suited and preferred for decarboxylating the C7-C11 2-ketoacids.

EXAMPLE 1

[0084] Design of A. brasilense Phenylpyruvate Decaroxylase (AbPPDC) Variants with Higher Catalytic Efficiency for 2-Ketononanoic Acid Decarboxylation

[0085] A crystal structure model of the ternary complex of AbPPDC with 3-deaza-thiamine diphosphate and 5-phenyl-2-oxovaleric acid (PDB ID Code 2Q5Q) is used to identify residues lining the 2-ketoacid binding pocket within the active site of AbPPDC. See, e.g., Versees, W.; Spaepen, S.; Wood, M. D.; Leeper, F. J.; Vanderleyden, J.; Steyaert, J. “Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase,” J. Biol. Chem., 2007, 282:35269-35278. The amino acid sites denominated as Met-380, Met-461, Phe-385, Phe-465, Gln-536 and Phe-532 are selected for substitution experimentation based on their relationship with 5-phenyl-2-oxovaleric acid. Substitutions of one or more sites are made as listed in Table 1 and the variants prepared.

[0086] Enzyme F532V replaces Phe-532 in AbPPDC with valine, while enzyme F532L replaces Phe-532 with leucine. Enzyme F385L/M461V replaces Phe-385 with leucine and Met-461 with valine. The remaining A. brasilense phenylpyruvate decarboxylase (AbPPDC) variants in the Table 1 are named according to the amino acid (first letter, with “F” representing “phenylalanine [Phe];” “M” representing “methionine” [Met]; and “Q” representing “glutamine” [Gln]), its position in the amino acid sequence (the number), and the amino acid used as a replacement (last letter, with “L” representing “leucine;” “V” representing “valine;” “A” representing “alanine”; “C” representing “cysteine”; “I” representing “isoleucine”; and “G” representing “glycine.”)

[0087] Each of the modified AbPPDC variants is expressed and purified, and then tested for activity against the three substrates, which are 2-ketohexanoate (2-KH), 2-ketooctanoate (2-KO) and 2-ketononanoate (2-KN). The 2-KH, 2-KO and 2-KN would be anticipated to form pentanal, heptanal and octanal, respectively, upon decarboxylation by AbPPDC.

[0088] The evaluation of the AbPPDC variants is performed in two steps using the high-throughput enzyme assay described hereinbelow. Initially, all the variants are tested for activity against a single high concentration (2 mM) of 2-KH and 2-KN (as shown in Table 1). Following the initial evaluation, detailed kinetic analysis is performed on a select number of variants to determine the maximal rate (k.sub.cat), substrate concentration yielding half maximal rate (K.sub.0.5, equivalent of K.sub.M for enzymes following Michaelis-Menten kinetics), and the catalytic efficiency of the enzyme (k.sub.cat/K.sub.0.5) against 2-KO and 2-KN (as shown in Table 2). AbPPDC variants, having higher specificity (higher k.sub.cat/K.sub.0.5) for 2-KN, will be efficient in producing octanal and chemicals derived from it inside the cells.

TABLE-US-00001 TABLE 1 Sequence listings and activity of AbPPDC variants Activity, nmol .Math. min.sup.−1 .Math. mg.sup.−1 Enzyme SEQ ID 2-KH 2-KN AbPPDC 4 4.5 ± 2.7  199 ± 18 M380V 6 9.4 ± 0.6  178 ± 17 F385L 8 11.9 ± 2.6   78 ± 2 F385V 10 9.6 ± 0.4 145 ± 3 F385I 12 0.8 ± 0.0  25 ± 0 M461C 14 17.7 ± 12.sup.   241 ± 14 M461V 16 9.6 ± 0.2  332 ± 22 M461L 18 .sup. 2 ± 0.8 293 ± 3 M461A 20 1.2 ± 0.1  40 ± 2 F465L 22 1.8 ± 6.3  99 ± 2 F532A 24 0.6 ± 0.1  6 ± 0 F532G 26 0.4 ± 0.1 106 ± 0 F532V 28 0.0 209 ± 3 F532L 30 0.0 309 ± 8 Q536G 32 1.1 ± 0.3 251 ± 1 Q536A 34 4.7 ± 0.9 396 ± 1 Q536L 36 53 ± 1  497 ± 7 Q536I 38 136 ± 2   715 ± 57 Q536V 40 57 ± 1   779 ± 16 F532V/Q536V 42  16 ± 0.1  307 ± 10 M380L/M461V 44 2.8 ± 1.4 156 ± 8 M380V/M461V 46 .sup. 4 ± 0.9 196 ± 8 F385V/M461V 48 5.9 ± 2.sup.   100 ± 15 F385L/M461V 50 8.1 ± 1.5  78 ± 1 F532A/Q536V 52 1.6 ± 0.2 238 ± 1 F532V/Q536A 54 1 ± 3 242 ± 0 F385L/Q536V 56 1.8 ± 0.4 164 ± 2 F385V/Q536V 58 3.5 ± 0.5 .sup. 219 ± 0.5 M461V/Q536V 60 11.8 ± 0.1  312 ± 2 M461L/Q536V 62 0   644 ± 5 M461A/Q536V 64 1.7 ± 0.1 272 ± 7 M461V/F532V 66 1.3 ± 0.3 260 ± 3 F465L/Q536V 68 2.6 ± 0.5 327 ± 1 F465V/Q536V 70 0.9 ± 0.1 201 ± 1 F465L/F532V 72 6.2 ± 0.4  393 ± 40 F532A/Q536A 74 1.4 ± 0.8  57 ± 6 M461V/F532V/Q536V 76 1.6 ± 2.5  494 ± 28 M380V/M461V/Q536V 78 0.0 195 ± 7 F385L/M461L/Q536V 80 1.4 ± 0.8 129 ± 1 M380V/F385V/M461V 82 6.7 ± 0.5  87 ± 7 SEQ ID 4 is the amino acid sequence of A. brasilense phenylpyruvate decarboxylase (GenBank: Accession No. L26240). SEQ ID 6-82 are sequences of proteins designed and expressed in this invention. All the proteins expressed in this invention have 13 additional amino acids at the N-terminus, added as the histidine-tag (shown in SEQ ID 2).

EXAMPLE 2

[0089] A. Heterologous Expression of Azospirillum brasilense Phenylpyruvate Decarboxylase (AbPPDC) and its Engineered Variants in E. coli

[0090] To evaluate the substrate specificity of the wild type AbPPDC and its variants listed in Table 1, genes of all the proteins are expressed in E. coli cells separately and the protein products are isolated from the cells. The gene sequence of the Azospirillum brasilense phenylpyruvate decarboxylase (GenBank: Accession no. L26240) is downloaded from the NCBI database. Codons of 13 additional amino acids that include six (6) histidines (his) are added upstream of the Met-1 codon of the AbPPDC gene sequence. Such a modification allows expression of a Histidine-tagged AbPPDC having 13 additional amino acids on the N-terminus. The additional amino acids are attached as an aid for purifying the protein in a single step using Ni-NTA chromatography. The entire AbPPDC sequence with 13 additional amino acids (SEQ ID 2) is chemically synthesized and then cloned into the pRSFDuet-1 vector (EMD Biosciences) downstream of the T7 polymerase promoter by Synthetic Genomics, Inc. (San Diego, Calif.). The final vector is sequenced by Synthetic Genomics, Inc. before shipping.

[0091] Genes of the AbPPDC variants listed in Table 1 are either chemically synthesized or generated using New England Biolab's Q5 Site-directed Mutagenesis Kit (cat. no. E0554S) and cloned into the pRSFDuet-1 vector. The pRSFDuet-1 vector containing AbPPDC or the AbPPDC variant gene is transformed into E. coli, AbPPDC or its variant, then expressed and eventually purified, as described below.

[0092] E. coli expression studies are then 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 AbPPDC expression vector are selected using kanamycin as the marker in the growth medium.

[0093] E. coli transformants harboring the AbPPDC or AbPPDC variant expression vector are then selected on Luria-Bertani (LB) broth agar plates containing 50 micrograms per milliliter (μg/mL) of kanamycin. The plates are incubated at 37 degrees Celsius (° C.) for 16 hours (h). A starter culture is started by transferring a single colony of transformant into 50 milliliters (mL) of LB medium containing 50 ug/mL of kanamycin and incubated at 37° C. with shaking at 220 revolutions per minute (rpm) overnight. On the 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 (OD.sub.600 nm) of 0.5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM is added to induce the expression of the AbPPDC or AbPPDC variant genes and the culture is transferred to a 15° C. incubator for 16 hours (h). At the end of 16 h, the culture is centrifuged at 8000 revolutions per minute (rpm) to pelletize the cells. The cell pellet is divided into two aliquots and stored at −80° C. overnight before purification.

[0094] An E. coli cell pellet taken from 400 mL of expression culture is suspended in B-PER reagent (Thermo Fisher Scientific, Inc., Rockford, Ill.) containing 1 μg/mL of DNAse (Thermo Fisher Scientific, Inc., Rockford, Ill.), 1 μg/mL of lysozyme (Thermo Fisher Scientific, Inc., Rockford, Ill.), 1 millimolar (mM) of dithiothreitol, and protease inhibitor cocktail (RPI Corp., Mount Prospect, Ill.). The suspension is rocked gently for 30 minutes (min) at room temperature and centrifuged at 15,000 times gravity (×g) for 20 min to pelletize cell debris. The supernatant is separated and incubated with 5 mL of Co-NTA resin (Thermo Fisher Scientific, Inc., Rockford, Ill.) 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% glycerol). Following an incubation period of 1 h at 4° C., the enzyme bound resin is washed with 5 volumes of equilibration buffer. AbPPDC or its variants are eluted from the Co-NTA resin with equilibration buffer containing 200 mM imidazole. The eluted proteins are dialyzed against phosphate buffered saline and stored as a 20% glycerol solution at −20° C.

B. Determination of the Substrate Specificity of AbPPDC and AbPPDC Variants

[0095] The evaluation of the substrate specificities of AbPPDC variants is performed using the methods as described in detail in Example 1.

[0096] A high-throughput AbPPDC coupled enzyme assay is developed for evaluating the substrate specificity of AbPPDC variants. The assay involves reducing the aldehyde produced from AbPPDC mediated 2-ketoacid decarboxylation, using an alcohol dehydrogenase (ADH6, GenBank: Accession No. NP 014051.3). The initial velocities of the AbPPDC catalyzed reactions are determined from the rates of oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) occurring during the ADH6 catalyzed reduction of aldehyde.

[0097] The HTP screening assay involves incubating 2 mM 2-KH or 2 mM 2-KN with 0.5 mM thiamine diphosphate, 0.35 mM NADPH, 4.7 micrograms (μg) of yeast ADH6 (GenBank: Accession No. NP 014051.3) and 0.3 milligrams per milliliter (mg/mL) bovine serum albumin (BSA) in AbPPDC assay buffer (50 mM 3-(N-morpholino)propanesulfonic acid, pH 6.8, containing 2.5 mM magnesium chloride (MgCl.sub.2)) at 30° C. The reaction is started by addition at 30° C. of working enzyme stock containing from 0.5 μg to 3.5 μg of AbPPDC variant diluted in AbPPDC assay buffer containing 1 mg/mL BSA. The plate containing the 200 μL of reaction mixture is centrifuged at 2500×g for 15 sec and the absorbance change of the reaction mixture followed spectrophotometrically at 340 nm on a BioTek™ plate reader, pre-equilibrated at 30° C. Initial velocity of the enzyme reaction is calculated using the rate of NADPH consumption at 340 nm and the extinction coefficient of NADPH (6.22 mM.sup.−1 cm.sup.−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 milligram (nmol.Math.min.sup.−1.Math.mg.sup.−1). Protein concentrations for normalizing the activities are determined using the 660 nm total protein assay kit from Pierce Biotechnology Inc., available from Thermo Fisher Scientific, Inc., using BSA as the standard.

[0098] The kinetic parameters of the decarboxylation of 2-ketooctanoate (2-KO) and 2-ketononanoate (2-KN) by AbPPDC and its variants are also determined using the same HTP AbPPDC coupled enzyme assay, except that the concentrations of 2-KO or 2-KN are varied from 0 to 4 mM.

[0099] For AbPPDC variants exhibiting substrate activation, as evident from a sigmoidal plot of initial velocities versus substrate concentration plot, the kinetic parameters (k.sub.cat, K.sub.0.5, and k.sub.cat/K.sub.0.5) of 2-keto-acid decarboxylation are obtained by fitting the data to the Hill equation (shown in the legend of Table 2) using nonlinear regression. For variants following normal saturation kinetics, the kinetic parameters (k.sub.cat, K.sub.M, and k.sub.cat/K.sub.M) are obtained by fitting initial velocities to the Michaelis-Menten equation using nonlinear regression. Nonlinear regression is performed using the GraphPad Prism™ software. Table 2 lists the kinetic parameters of 2-KO and 2-KN decarboxylation by AbPPDC and its variants. The amount of enzyme in the reaction mixture is determined using the Pierce Biotechnology InC.™ 660 nm total protein assay kit and using BSA as the standard.

[0100] Narrowing the substrate specificity of AbPPDC is expected to improve the accumulation of a specific aldehyde and its downstream products. In general AbPPDC prefers bulkier 2-ketoacids, such as 5-phenyl-2-ketopentanoate and phenylpyruvic acid, as evidenced by high catalytic efficiencies with respect to those substrates (See, e.g., Spaepen, S.; Versees, W.; Gocke, D.; Pohl, M.; Steyaert, J.; Vanderleyden, J. “Characterization of phenylpyruvate decarboxylase, involved in auxin production of Azospirillum brasilense,” J. Bacteriol., 2007, 189:7626-7633).

[0101] AbPPDC and the variants listed in Table 1 are screened for activity against 2 mM 2-ketohexanoate (2-KH) and 2 mM 2-ketononanoate (2-KN) as substrates. That screening reveals that the wild type AbPPDC catalyzes the decarboxylation of 2-KN, but exhibits poor activity against 2-KH under the assay conditions. All of the AbPPDC variants, also catalyze the decarboxylation of 2-KN, and exhibit relatively low activity against 2-KH (Table 1). Substitution of Gln-536 with alanine, valine, isoleucine or leucine increases the 2-KN decarboxylating activity over that of the wild type enzyme, but also improves activity against 2-KH as a substrate. These results suggest that all of the AbPPDC variants listed in Table 1 can be expressed in an active form in heterologous systems. Furthermore, all of them have significantly higher activity against 2-KN than 2-KH, suggesting that AbPPDC and the variants described herein prefer >C6 2-ketoacids.

[0102] Detailed steady state kinetic analysis is performed on all the enzymes to determine the maximal rate and the catalytic efficiency of decarboxylating 2-ketooctanoate (2-KO) and 2-ketononanoate (2-KN). Both the substrates exhibit hyperbolic and non-hyperbolic kinetics as evident from Table 2. For AbPPDC variants showing non-hyperbolic kinetics, initial velocities of the decarboxylations of 2-KO and 2-KN are fitted to the Hill equation (Table 2 legend) and the maximal rate and the catalytic efficiencies (k.sub.cat/K.sub.0.5) calculated as shown in Table 2. A Hill coefficient greater than 1 suggests presence of substrate activation with 2-KO and 2-KN. Substrate activations have been reported with AbPPDC and with other decarboxylases. See, also, Spaepen, S., Ibid.

[0103] As evident from Table 2, the amino acid substitutions affect the catalytic efficiency of the variants in capturing 2-KO and 2-KN for catalysis in different ways. For some variants, for example, F532V, the catalytic efficiency of decarboxylation of 2-KN and 2-KO is 180% and 45%, respectively, in comparison with that of the wild type AbPPDC. This suggests that F532V substitution increases the substrate specificity for 2-KN while decreasing it for 2-KO. The preference of the AbPPDC variants for 2-KN over 2-KO is calculated by taking the ratio of the variant's catalytic efficiencies and is shown in Table 2. As evidenced in Table 2, the specificities of AbPPDC and F532V are 1.8 and 5.6, respectively, indicating that their catalytic efficiency of decarboxylating 2-KN is 1.8 and 5.6 times higher than that of decarboxylating 2-KO. This also indicates that the F532V variant is 3-fold more specific than AbPPDC in preferring 2-KN over 2-KO. Similarly, the preference of F385L for 2-KN over 2-KO is 5-fold higher than that of AbPPDC. This data suggests that the F385L and F532V substitutions improve the substrate specificity for a longer 2-ketoacid (for example 2-KN) over shorter one (for example 2-KO). Thus, the F385L and F532V variants would improve the accumulation of longer (C7-C10) aldehyde based products when 2-ketoacids are being elongated using the “+1 pathway” (FIG. 1).

[0104] Similarly, the specificities of the M461L, F532L, Q536G, Q536L, F532V/Q536V, M380V/M461V, F532A/Q536V, F532V/Q536A, F385L/Q536V, M461V/F532V and M461V/F532V/Q536V variants for 2-KN, in comparison with the specificity of each variant for 2-KO, are 3.3, 4.3, 4.8, 2.7, 3.6, 2.7, 6.8, 4.6, 4.3, 5.4, and 2.1, respectively. This suggests that all of these variants are more specific than AbPPDC in capturing 2-KN for catalysis.

[0105] In addition to the specificity of the AbPPDC variant for 2-KN, maximal accumulation of octanal and biochemicals derived from it will also be dependent on the relative efficiencies of the 2-KN producing pathways versus that of the AbPPDC variant. For example, where the efficiency of the engineered 2-ketoacid chain extension pathway (involving the three enzymes, isopropylmalate synthase, isopropylmalate isomerase and isopropylmalate dehydrogenase) in producing 2-KN is relatively low compared to that for producing 2-KO, heptanal formation would result, due to the decarboxylation of 2-KO by AbPPDC variants in combination with reduction in the accumulation of octanal based chemicals inside the cells. Under such circumstances, AbPPDC variant such as F385L would be preferred decarboxylase based upon its relatively high specificity (9.1), coupled with its reduced efficiency as 2-KN decarboxylating catalyst (Table 2).

[0106] The results also show that substituting Gln-536 with a hydrophobic amino acid (i.e., glycine, alanine, valine, leucine, or isoleucine) improves the catalytic efficiency of AbPPDC and other specificity enhancing substitutions as shown in Table 2. The Q536V variant is 8- and 5.7-fold more efficient than the wild type enzyme in decarboxylating 2-KO and 2-KN, respectively (Table 2). Similarly, the M461V/F532V/Q536V variant is 27- and 10-fold more efficient than M461V/F532V variant in decarboxylating 2-KO and 2-KN, respectively (Table 2). The M461V/F532V/Q536V variant is about 17- and 20-fold more efficient enzyme than the wild type enzyme in decarboxylating 2-KO and 2-KN, respectively (Table 2). The higher catalytic efficiency of the M461V/F532V/Q536V variant allows effective decarboxylation of 2-KO at 17-fold lower intracellular levels than the wild type enzyme and promotes accumulation of heptanal-derived chemicals, such as heptanol (through coexpression with an alcohol dehydrogenase) or heptanoate (through coexpression of an aldehyde dehydrogenase) inside the cells.

[0107] Other substitutions of Gln-536, such as with glycine, alanine, leucine or isoleucine, which also improve the catalytic efficiency of decarboxylation, will also improve the catalytic efficiencies of specificity-enhancing substitutions. This is exhibited by Q536A substitution, which, when added into a F532V variant (with k.sub.cat/K.sub.0.5=4.8 mM.sup.−1 min.sup.−1 for 2-KO and k.sub.cat/K.sub.0.5=27 mM.sup.−1 min.sup.−1 for 2-KN), gives rise to a F532V/Q536A variant (with k.sub.cat/K.sub.0.5=8.3 mM.sup.−1 min.sup.−1 for 2-KO and k.sub.cat/K.sub.0.5=38 mM.sup.−1 min.sup.−1 for 2-KN) having 72% and 40% higher catalytic efficiencies, respectively, against 2-KO and 2-KN.

[0108] In summary, results suggest that the expression of AbPPDC and its genetically modified variants allow efficient decarboxylation of C7-C11, and particularly C7-C9 in this example, 2-ketoacids in vivo, and thereby allow accumulation of, for example, chemicals derived from aldehydes such as hexanal, heptanal, and/or octanal, inside the cells. Furthermore, modifications of F532, F385, Q536, M380, M461, F465 either alone or in combination, may give rise to microbial organisms that exhibit specifically improved accumulation of, for example, similarly-derived chemicals inside the cells.

TABLE-US-00002 TABLE 2 Kinetic characterization of AbPPDC and its variants* 2-ketooctanoate (2-KO) 2-ketononanoate (2-KN) k.sub.cat, k.sub.0.5 or K.sub.M, k.sub.cat/K.sub.0.5, kcat, K.sub.0.5 or K.sub.M, k.sub.cat/K.sub.0.5, Description min.sup.−1 mM h mM.sup.−1 .Math. min.sup.−1 min-1 mM h mM.sup.−1 .Math. min.sup.−1 Spec AbPPDC  15 ± 0.7 1.7 ± 0.1 2.4 ± 0.1 9.0 ± 0.7 20 ± 0 1.23 ± 0.02 2.1 ± 0.1 16.2 ± 0.4 1.8 .sup.1F385L 1.7 ± 0.3 2.3 ± 0.5 2.3 ± 0.8 0.8 ± 0.2 17 ± 1 2.39 ± 0.26 2.7 ± 0.4  7.0 ± 1.0 9.1 M461L  12 ± 0.5 1.4 ± 0.1 2.6 ± 0.4 8.9 ± 0.7 28 ± 1 0.97 ± 0.04 2.1 ± 0.1   29 ± 1.4 3.3 F532V 9.8 ± 0.2 2.0 ± 0.1 3.4 ± 0.3 4.8 ± 0.2 23 ± 2 0.89 ± 0.15 1.8 ± 0.4 27 ± 5 5.6 F532L  13 ± 0.3 1.6 ± 0.1 2.5 ± 0.2 8.2 ± 0.3 30 ± 5 0.94 ± 0.27 1.4 ± 0.3  35 ± 12 4.3 Q536G  11 ± 0.8 1.9 ± 0.2 2.2 ± 0.2 5.8 ± 0.6 21 ± 0 0.76 ± 0.02 2.7 ± 0.2 28 ± 1 4.8 Q536A  20 ± 0.2 0.72 ± 0.02 2.1 ± 0.1 27 ± 1  30 ± 1 0.52 ± 0.03 1.6 ± 0.1 58 ± 4 2.1 Q536L.sup.§  27 ± 1.7 1.34 ± 0.23 — 21 ± 4  83 ± 6 1.51 ± 0.26 —  56 ± 11 2.7 Q536I.sup.§  32 ± 1.1 0.71 ± 0.07 — 45 ± 5  63 ± 3 0.69 ± 0.09 —  92 ± 13 2.0 Q536V  39 ± 1.3 0.55 ± 0.05 1.3 ± 0.2 72 ± 6  41 ± 3 0.45 ± 0.08 1.2 ± 0.3  93 ± 18 1.3 F532V/Q536V  16 ± 0.3 0.42 ± 0.01 4.3 ± 0.4 39 ± 1  21 ± 1 0.15 ± 0.01 5.3 ± 0.8 141 ± 7  3.6 M380V/M461V.sup.§ 3.1 ± 0.1 0.18 ± 0.02 — 17 ± 2    6 ± 0.1 0.13 ± 0.02 — 46 ± 6 2.7 F385L/M461V.sup.§ 2.8 ± 0.1 0.61 ± 0.09 — 4.7 ± 0.7 12 ± 1 3.47 ± 0.53 —  3.6 ± 0.7 0.7 F532A/Q536V 9.1 ± 0.7  2.0 ± 0.15 2.9 ± 0.3 4.5 ± 0.5 17 ± 0 0.54 ± 0.02 3.7 ± 0.5 31 ± 1 6.8 F532V/Q536A  11 ± 0.2  1.3 ± 0.03 1.3 ± 0.0  8.3 ± 0.24 19 ± 1 0.49 ± 0.03 3.1 ± 0.4 38 ± 2 4.6 F385L/Q536V 5.4 ± 1.1  2.4 ± 0.52 2.0 ± 0.3 2.4 ± 0.7 15 ± 1 1.46 ± 0.05 2.7 ± 0.2 10 ± 1 4.3 M461L/Q536V 57 ± 2   1.2 ± 0.10 1.4 ± 0.1 49 ± 5  65 ± 5 0.72 ± 0.11 1.3 ± 0.2  92 ± 16 1.9 M461A/Q536V.sup.§ 45 ± 3   1.7 ± 0.25 — 28 ± 4  54 ± 6 2.02 ± 0.41 — 28 ± 7 1.0 M461V/F532V  12 ± 1.1 2.2 ± 0.3 1.9 ± 0.2 5.6 ± 0.8 32 ± 2 1.05 ± 0.09 1.8 ± 0.2 31 ± 3 5.4 F465L/Q536V  25 ± 0.5 0.29 ± 0.02 2.0 ± 0.2 85 ± 5  29 ± 1 0.18 ± 0.01 1.9 ± 0.3 164 ± 11 1.9 M461V/F532V/Q536V 65 ± 2  0.43 ± 0.03 2.1 ± 0.3 152 ± 13  43 ± 3 0.14 ± 0.02 2.8 ± 0.8 322 ± 47 2.1 M380V/M461V/Q536V.sup.§ 4.9 ± 0.2 0.62 ± 0.1  — 8.2 ± 1.3 25 ± 3 3.10 ± 0.73 —  8.6 ± 2.3 1.1 F385L/M461L/Q536V.sup.§ 3.4 ± 0.1 0.63 ± 0.08 — 5.5 ± 0.7 15 ± 1 2.65 ± 0.49 —  6.1 ± 1.2 1.1 *Initial velocity studies are determined using the HTP coupled assay described in the text. The initial velocities of [00001]$\mathrm{all}.Math..Math.\mathrm{the}.Math..Math.\mathrm{enzymes}.Math..Math.\mathrm{except}.Math..Math.\mathrm{those}.Math..Math.\mathrm{indicated}.Math..Math.\mathrm{by}.Math..Math.\S .Math..Math.\mathrm{are}.Math..Math.\mathrm{fitted}.Math..Math.\mathrm{to}.Math..Math.\mathrm{the}.Math..Math.\mathrm{Hill}.Math..Math.\mathrm{equation}.Math..Math.(v=\frac{\mathrm{kcat}.Math.s^h}{K_{0.5}^h+s^h};$v is the initial velocity at a given substrate concentration, S) using the GraphPad Prism ™ software. k.sub.cat, K.sub.0.5, h and k.sub.cat/K.sub.0.5 are the maximal velocity, substrate concentration at half the maximal velocity, Hill coefficient and catalytic efficiency respectively. Results are the mean ± standard error of 2-3 independent experiments. Specificity of the AbPPDC variant is calculated by taking the ratio of catalytic efficiencies (k.sub.cat/K.sub.0.5) of the variant for 2-KN to that for 2-KO. .sup.1The applied naming convention is that the first letter indicates the amino acid residue which has been altered. F = phenylalanine [Phe]; Q = glutamine [Gln]; M = methionine [Met]. The number indicates the position in the amino acid sequence (shown are positions 380, 385, 461, 465, 532, and 536, accordingly). The last letter indicates the amino acid residue that is substituted at that location. G = glycine; A = alanine; I = isoleucine; V = valine; L = leucine. .sup.§Initial velocities of these variants are fitted to the classical Michaelis-Menton equation.

EXAMPLE 3

[0109] In Vitro Synthesis of C5-C9 Alcohols with the F385L Variant (SEQ ID. 8) of Azospirillum brasilense Phenylpyruvate Decarboxylase (AbPPDC)

[0110] In vitro synthesis of linear alcohols with the F385L variant is performed by incubating 0.5 mM 2-ketobutyrate (2-KB) with 0.5 mM thiamine diphosphate, 2.5 mM NAD.sup.+, 0.2 milligrams per milliliter (0.2 mg/mL) bovine serum albumin (BSA), 5 mM acetyl coenzyme A, 0.036 mg/mL of the H97A/S139G/N167G/P169A/G462D variant of E. coli isopropylmalate synthase (reported by Marcheschi, R. J., et al. “A Synthetic Recursive “+1” pathway for carbon chain elongation” ACS Chem. Biol. 7:689-697, 2012), 0.16 mg/mL of LeuC subunit of isopropylmalate isomerase (GenBank Accession No. NC 000913.3 Gene ID: 945076) and 0.21 mg/mL of LeuD subunit of isopropylmalate isomerase (GenBank Accession No. NC 000913.3 Gene ID: 945642), 0.264 mg/mL of E. coli isopropylmalate dehydrogenase (LeuB; GenBank Accession No. NC_000913.3 Gene ID: 944798), 0.192 mg/mL of L96G/V198A variant of isopropylmalate dehydrogenase (reported in WO2015089127 A1), 0.025 mg/mL of Saccharomyces cerevisiae alcohol dehydrogenase (ADH6, GenBank: Accession No. NP_014051.3) and 0.0054 mg/mL of F385L variant (SEQ ID 8) in in vitro synthesis buffer (50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, pH 7.5, containing 30 mM potassium chloride (KCl) and 5 mM magnesium chloride (MgCl.sub.2)).

[0111] The reaction is initiated with the addition of 2-ketobutyrate to the rest of the reaction mixture. An equal volume of analytical grade toluene (CHROMOSOLVPlus™ for HPLC, ≧99%, catalog number 650579) is overlaid on top of the reaction mixture and the solution is incubated at 30° C. NADPH is added to the aqueous layer to a final concentration of 1 mM after 2.5 hours of incubation at 30° C. Additional NADPH is added to the aqueous layer to a final concentration of 2 mM after 6 hours of incubation at 30° C. The reaction is incubated an additional 18 hours at 30° C., then stopped by freezing at −20° C. for 30 minutes. Part of the toluene layer is removed and analyzed using a Gas Chromatograph equipped with a Flame Ionization Detector (FID).

[0112] In vitro synthesis of branched alcohols with the F385L variant is performed by replacing 2-ketobutyrate with 0.5 mM 2-ketoisovalerate (2-KIV) or 0.5 mM 3-methyl-2-ketopentanoate (3M-2KP) in the above reaction mixture and performing the experiment as described above.

[0113] Alcohols are quantified using a Hewlett Packard (HP) 6890 Series Gas Chromatograph equipped with a Flame Ionization Detector (FID), a model G1513A automatic injector, and a GC AutoSampler Controller. The analytes are separated using an Agilent J&W DB-FFAP capillary GC column (30 m×0.320 mm ID×0.25 μM film thickness; catalog number 123-3232, Agilent Technologies, Inc., Wilmington, Del. 19808). The initial GC oven temperature is 40° C., which is held for 1.50 minutes, then is increased to 235° C. with a 40° C./minute gradient. This gradient gives a total run time of 6.38 minutes. The column flow rate is 4.0 mL/minute, with helium as the carrier gas. The injection volume is 1 μL. The temperature settings for the injector and detector are 225° C.

[0114] The alcohol titers produced from these in vitro synthesis reactions are shown in FIGS. 2, 3 and 4. The results indicate that the F385L variant, in combination with the H97A/S139G/N167G/-P169A/G462D variant of E. coli isopropylmalate synthase (LeuA), E. coli isopropylmalate isomerase (LeuCD), wild type and modified E. coli isopropylmalate dehydrogenase (LeuB), and alcohol dehydrogenase (ADH6), produces elongated C5-C9 alcohols upon incubation with 2-ketobutyrate, 2-ketoisovalerate, or 3-methyl-2-ketopentanoate. Moreover, the results demonstrate the specificity of the F385L variant for longer linear alcohols, wherein 1-octanol represents approximately 60% of the total alcohols generated upon incubation with 2-ketobutyrate. With branched chain 2-ketoacids (2-ketoisovalerate, KIV, and 3-methyl-2-ketopentanoate, 3M-2-KP), approximately equivalent amounts of 5-methyl-1-hexanol and 6-methyl-1-heptanol are produced upon incubation with 2-ketoisovalerate, and approximately equivalent amounts of 3-methyl-1-pentanol and 5-methyl-1-heptanol are produced upon incubation with 3-methyl-2-ketopentanoate. These results demonstrate that the F385L variant accepts linear as well as branched chain 2-ketoacids as substrates and can produce corresponding linear and branched chain aldehydes which could subsequently be converted to other products such as alcohols, carboxylic acids, or alkanes.

EXAMPLE 4

[0115] In Vivo Production of C5-C8 Alcohols in Engineered Strains of E. Con Using Wild Type AbPPDC and its Variants in Combination with the “+1 Pathway” Enzymes

[0116] Escherichia coli (E. coli) MG1655 is engineered to promote long-chain linear alcohol production and to enable gene expression from a T7 promoter. To improve linear alcohol production, ilvBN and ilvIH are inactivated via λRed-mediated homologous recombination as described by Datsenko, K A, Wanner, B L, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products,” Proc. Natl. Acad. Sci. U.S.A. 2000, 97(12), 6640-6645. The ilvBN and IlvIH genes are involved in branched chain amino acid production, so the inactivation of these genes eliminates the production of branched chain alcohols. The ilvA gene, which is involved in the production of 2-ketobutyrate, is upregulated by replacing its native promoter and ribosome binding site with a strong constitutive promoter and strong ribosome binding site via λRed-mediated homologous recombination as described by Datsenko and Wanner, Ibid. To enable the expression of genes from T7 promoters, the DE3 lysogen is integrated into MG1655 using the ΔDE3 Lysogenization Kit (EMD Millipore Cat #69734). The resulting strain genotype is MG1655(DE3) ΔilvBN ΔilvIH ilvAup.

[0117] C5-C8 alcohols are produced in the engineered E. coli strain through the expression of eight proteins: (1) E. coli isopropylmalate synthase (LeuA); (2) engineered isopropylmalate synthase (described by Marcheschi, et al. ACS Chem. Biol. 2012, 7, 689-697); (3) and (4) two subunits of E. coli isopropylmalate isomerase (LeuCD); (5) isopropylmalate dehydrogenase (LeuB); (6) L96G/V198A variant of E. coli isopropylmalate dehydrogenase (as described in greater detail in co-pending International Patent Application Serial No. PCT/US14/69438, filed Dec. 10, 2014 (Attorney Docket No. 75413-WO-PCT), claiming the benefit of U.S. Provisional Patent Application No. 61/915,040, filed Dec. 12, 2013 (Attorney Docket No. 75413-US-PSP), both of which are incorporated herein in their entireties by reference); (7) AbPPDC or its variants; and (8) S. cerevisiae alcohol dehydrogenase (ADH6). Eleven strains are created in total. One strain is created containing only wild type AbPPDC. As a negative control, a strain with no PPDC is also created. Eight strains containing AbPPDC variants F532V, F358L, F385V, F532V Q536V, M461C, M461V, F385V M461C and F385L M461V are also created. Lastly, a strain containing wild type Lactococcus lactis keto-isovalerate decarboxylase (KIVD; Gene Accession No. AJ746364) is created as a comparison, as prior work has shown that KIVD is capable of producing long-chain alcohols in combination with the “+1 pathway” enzymes. See, e.g., Marcheschi, et al. Ibid.

[0118] The Novagen Duet Vector system (EMD Millipore Cat #71146, 71341, 71340, and 71147), which allows for the simultaneous expression of eight genes using four compatible plasmids, is used to express the genes mentioned above. Each of the four Duet vectors is cloned with two of the eight genes downstream of T7 promoters, and the four Duet vectors are transformed into the engineered E. coli strain. Recombinant strains bearing all of the plasmids are selected for using antibiotics (ampicillin at 25 micrograms per milliliter, μg/mL, chloramphenicol at 17 μg/mL, spectinomycin at 25 μg/mL, and kanamycin at 15 μg/mL) and confirmed with polymerase chain reaction (PCR) using methods known to those skilled in the art. Antibiotics are added at each solid and liquid cultivation step to ensure maintenance of the plasmids. After transformation, plate selection and PCR confirmation, strains are initially cultivated on a Luria-Bertani (LB) agar plate grown at 37° C. A single agar plate colony is used to inoculate 50 mL of LB medium in a 250 mL shake flask which is cultivated aerobically at 37° C. using an incubator shaker set at 200 rpm.

[0119] After 12-16 hours of cultivation in the LB shake flasks, serum bottles are inoculated at 1% v/v to evaluate alcohol production. Serum bottle fermentation medium is prepared using deionized water according to the concentrations shown in Table 1. The medium is filter sterilized, and 20 mL of medium is added to butyl rubber-stoppered 125 mL serum bottles. Prior to media addition, serum bottles are pre-sterilized by autoclaving at 125° C. for 30 minutes using a Steris Amsco Century SV-160H Prevac Sterilizer.

TABLE-US-00003 TABLE 1 Medium composition used to demonstrate alcohol production from E. coli recombinantly engineered to contain the “+1 pathway” in combination with Azospirllum brasilense decarboxylase (AbPPDC) or its variants. Component Concentration (g/L) MOPS buffer 26.2 Glycerol 20 Tryptone 10 Yeast Extract 5 Calcium pantothenate 1.19 Na2HPO4 0.105 (NH4)2SO4 0.661 NH4Cl 1.6

[0120] After inoculation, serum bottle cultures are cultivated at 37° C. with shaking at 200 rpm in an incubator shaker. Approximately three hours after inoculation, the cultures are induced using 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) to ensure expression of all genes. Fermentations are harvested for analysis 24 hours after induction.

[0121] At the end of the fermentation, serum bottles are immediately chilled to 4° C. by placing in a refrigerator for 20-30 minutes. Serum bottles are de-capped, and the fermentation broth is quickly poured into a 50 mL conical tube containing 1 mL of a saturated sodium chloride solution and 2 mL of analytical grade toluene (CHROMOSOLV PIus™ for HPLC, ≧99.9%, catalog number 650579). The broth-sodium chloride-toluene mixture is vortexed for 30 seconds. A 300 μL aliquot of the toluene extract is then submitted for analysis using GC/FID as described in Example 3.

[0122] The mean alcohol distributions for the serum bottles are shown in FIG. 5. The results indicate that expression of the wild type Azospirllum brasilense decarboxylase (AbPPDC) in combination with the “+1 pathway” genes and ADH6 results in a functional pathway for the production of linear alcohols ranging from pentanol to octanol. No C5-C8 alcohols are detected in strains without AbPPDC, confirming that the presence of this gene is essential for long-chain alcohol production. Furthermore, the results demonstrate that the strain containing wild type AbPPDC accumulated substantially more hexanol, heptanol and octanol than the strain containing KIVD. No hexanol, heptanol or octanol production is detected in the KIVD strain, but the AbPPDC WT strain produces >2 mg/L, >3 mg/L, and >0.1 mg/L of hexanol, heptanol and octanol, respectively. Approximately 50% of alcohol production is heptanol and octanol, which is a significant improvement compared to previous work with other decarboxylases that result primarily in pentanol and hexanol production (Marcheschi, et al. ACS Chem. Biol. 2012, 7:689-697). Thus, the use of the AbPPDC decarboxylase appears to shift alcohol production to longer chain lengths, a result which is consistent with the in vitro data contained within Examples 1 and 2.

[0123] The additional data in FIG. 5 demonstrates that all of the AbPPDC variants have the ability to produce C5-C8 alcohols. Three of the AbPPDC variants, M461C, M461V and F385L/M461V, demonstrate a significant improvement in C5-C8 alcohol production compared to the wild type AbPPDC. AbPPDC variant M461C produces >3 mg/L of hexanol and >5 mg/L of heptanol, representing more than a 40% improvement compared to the wild type AbPPDC. Most impressively, AbPPDC variant M461V shows more than 2-fold improvements in pentanol, hexanol and heptanol production compared to wild type AbPPDC. The M461V variant also shows a 30% improvement in terms of octanol titer relative to the wild type AbPPDC. This strain containing the M461V variant produces the highest titers with ˜9 mg/L of hexanol and ˜8.5 mg/L of heptanol. Variants F532V, F385L, F532V Q536V, M461C, M461V and F385L M461V all show improvements in 1-octanol titer compared to KIVD and the AbPPDC wild type gene. Lastly, AbPPDC variant F385L/M461V shows about 60% improvement in hexanol and heptanol production compared to the AbPPDC wild type.