NZ622451B2 - Microorganisms and methods for producing alkenes - Google Patents

Abstract

Non-naturally occurring microbial organisms are disclosed containing an alkene pathway having at least one exogenous nucleic acid encoding an alkene pathway enzyme expressed in a sufficient amount to convert an alcohol to an alkene. The disclosure additionally provides methods of using such microbial organisms to produce an alkene, by culturing a non-naturally occurring microbial organism containing an alkene pathway as described herein under conditions and for a sufficient period of time to produce an alkene. l organisms to produce an alkene, by culturing a non-naturally occurring microbial organism containing an alkene pathway as described herein under conditions and for a sufficient period of time to produce an alkene.

Description

MICROORGANISMS AND METHODS FOR PRODUCING ALKENES BACKGROUND OF THE INVENTION This application claims the benefit of priority of United States Provisional application serial No. 61/535,893, filed September 16, 2011, the entire contents of which is incorporated herein by reference.

The present ion relates lly to biosynthetic processes, and more specifically to organisms having an alkene biosynthetic capability.

Alkenes are commonly produced by cracking the alkanes found in crude oil.

Cracking uses heat and a catalyst to decompose s. lly, alkenes are unsaturated hydrocarbons with one double bond (R-C=C-R). e of to the inherent property of alkenes being more reactive than alkanes due to the presence of a double bond, alkenes are ntly used in the manufacture of plastics. For example, alkenes are used in the maufacutre of polyethene, nylchloride (PVC) and Teflon. Lower s, which are obtained by the cracking of kerosene or , are also commonly used as fuel and illuminant. Some alkenes, such as 1,3-butadiene, styrene and propylene, are particularly useful in manufacturing.

Over 25 n pounds of butadiene (l,3-butadiene, BD) are produced annually and is applied in the manufacture of polymers such as synthetic s and ABS resins, and chemicals such as hexamethylenediamine and l,4-butanediol. Butadiene is typically produced as a by-product of the steam cracking process for conversion of petroleum feedstocks such as naphtha, liquefied petroleum gas, ethane or natural gas to ethylene and other olefins. The ability to manufacture butadiene from alternative and/or renewable ocks would represent a major advance in the quest for more sustainable chemical production processes

[0005] One possible way to produce butadiene renewably involves fermentation of sugars or other feedstocks to produce diols, such as l,4-butanediol or l,3-butanediol, which are separated, purified, and then dehydrated to butadiene in a second step involving metal-based catalysis. Direct fermentative production of butadiene from renewable feedstocks would obviate the need for dehydration steps and butadiene gas (bp -4.4°C) would be uously d from the fermenter and readily condensed and collected.

Developing a fermentative production process would eliminate the need for fossil-based butadiene and would allow substantial savings in cost, energy, and harmful waste and ons relative to petrochemically-derived butadiene.

Styrene is the precursor to yrene and numerous copolymers. Styrene based ts include, acrylonitrile l,3-butadiene styrene (ABS), styrene-l,3-butadiene (SBR) rubber, styrene-l,3-butadiene latex, SIS (styrene-isoprene-styrene), S-EB-S (styrene-ethylene/butylene-styrene), styrene-divinylbenzene (S-DVB), and unsaturated polyesters. These materials are used in rubber, plastic, insulation, fiberglass, pipes, automobile and boat parts, food containers, and carpet backing.

[0007] Styrene is most commonly produced by the catalytic dehydrogenation of ethylbenzene. Ethylbenzene is mixed in the gas phase with 10—15 times its volume in high-temperature steam, and passed over a solid catalyst bed. Most ethylbenzene dehydrogenation catalysts are based on iron(III) oxide, promoted by several percent potassium oxide or potassium carbonate. Steam serves several roles in this on. It is the source of heat for powering the ermic reaction, and it removes coke that tends to form on the iron oxide st through the water gas shift on. The potassium er enhances this decoking reaction. The steam also dilutes the reactant and products, shifting the position of chemical equilibrium towards products. A typical styrene plant consists of two or three reactors in series, which operate under vacuum to enhance the conversion and selectivity. Typical per-pass conversions are ca. 65% for two reactors and 70-75% for three reactors.

Propylene is ed primarily as a by-product of petroleum refining and of ethylene production by steam cracking of hydrocarbon feedstocks. Propene is separated by fractional lation from hydrocarbon es obtained from cracking and other refining processes. Typical hydrocarbon feedstocks are from non-renewable fossil fuels, such as petroleum, l gas and to a much lesser extent coal. Over 75 billion pounds of propylene are manufactured ly, making it the second largest fossil-based chemical produced behind ne. Propylene is a base chemical that is converted into a wide range of polymers, r intermediates and chemicals. Some of the most common derivatives of al and polymer grade propylene are polypropylene, acrylic acid, butanol, butanediol, acrylonitrile, propylene oxide, isopropanol and cumene. The use of the propylene derivative, polypropylene, in the production of plastics, such as injection moulding, and fibers, such as carpets, accounts for over one-third ofUS. consumption for this derivative. Propylene is also used in the production of synthetic rubber and as a propellant or component in aerosols.

The ability to manufacture propylene from alternative and/or renewable feedstocks would ent a major advance in the quest for more sustainable al production processes. One possible way to e propylene renewably involves fermentation of sugars or other ocks to produce the alcohols 2-propanol (isopropanol) or l-propanol, which is ted, purified, and then dehydrated to propylene in a second step involving metal-based catalysis. Direct fermentative production of propylene from renewable ocks would obviate the need for dehydration. During fermentative production, propylene gas would be continuously emitted from the fermenter, which could be readily collected and condensed. Developing a fermentative tion process would also eliminate the need for -based propylene and would allow substantial savings in cost, energy, and harmful waste and emissions relative to petrochemically-derived propylene.

[0010] Thus, there exists a need for alternative methods for effectively producing commercial quantities of alkenes. The present ion satisfies this need and provides related advantages as well.

SUMMARY OF INVENTION The invention provides non-naturally occurring microbial organisms containing an alkene pathway having at least one exogenous nucleic acid encoding an alkene pathway enzyme expressed in a sufficient amount to convert an alcohol to an alkene. In some aspects of the invention, the microbial organism comprises an alkene pathway selected from: (1) an alcohol kinase and a ate lyase; (2) a diphosphokinase and a phate lyase; and (3) an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase. The invention additionally provides methods of using such microbial organisms to produce an alkene, by culturing a non-naturally ing microbial organism containing an alkene pathway as described herein under conditions and for a sufficient period of time to produce an alkene.

BRIEF PTION OF THE DRAWINGS Figure 1 shows the conversion of an alcohol substrate to an alkene Via alkyl phosphate or alkyl diphosphate intermediates. Enzymes are A. l kinase, B. phosphate lyase, C. diphosphokinase, D. alkyl phosphate kinase and E. diphosphate lyase.

R1, R2, R3, and R4 are each ndently (a) hydrogen, cyano, halo, or nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl, each optionally substituted with one or more substituents Q; or (c) —C(O)R1a, —C(O)OR1a, —C(O)NR1bR1°, —C(NR1a)NR1bR1°, —0R1a, —OC(O)R1a, —OC(O)OR1a, —OC(O)NR1bR1°, —OC(=NR1a)NR1bR1°, R1a, —OS(O)2R1a, —OS(O)NR1bR1°, —OS(O)2NR1bR1°, 1°, —NR1aC(O)R1d, —NR1aC(O)OR1d,—NR1aC(O)NR1bR1°, —NR1aC(=NR1d)NR1bR1°u —NR1aS(O)Rld, —NR1aS(O)2R1d, —NR1aS(O)NR1bR1°, —NR1aS(O)2NR1bR1°, —SR1a, — S(O)R1a, —S(O)2R1a, —S(O)NR1bR1°, or —S(O)2NR1bR1°; n each R”, Rlb, R”, and R1d is independently en, C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 lkyl, C644 aryl, C745 aralkyl, heteroaryl, or cyclyl; or R1&1 and R10 together with the C and N atoms to which they are attached form heterocyclyl; or R1b and R10 together with the N atom to which they are attached form heterocyclyl; wherein each Q is independently selected from (a) oxo, cyano, halo, and nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 l, heteroaryl, and heterocyclyl, each of which is further optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; and (c) —C(O)Ra, —C(O)ORa, —C(O)NRbR°, —C(NRa)NRbR°, —0Ra, —OC(O)Ra, —OC(O)ORa, —OC(O)NRbR°, —OC(=NRa)NRbR°, —OS(O)Ra, —OS(O)2Ra, —OS(O)NRbR°, —OS(O)2NRbR°, —NRbR°, —NRaC(O)Rd, —NRaC(O)ORd, —NRaC(O)NRbR°, —NRaC(=NRd)NRbR°, —NRaS(O)Rd, —NRaS(O)2Rd, —NRaS(O)NRbR°, —NRaS(O)2NRbR°, —SRa, a, —S(O)2Ra, — S(O)NRbR°, and —S(O)2NRbR°, wherein each Ra, Rb, RC, and Rd is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl, each optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; or (iii) Rb and Rc together with the N atom to which they are attached form heterocyclyl, optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa;wherein each Qa is independently selected from the group consisting of (a) oxo, cyano, halo, and nitro; (b) C1- 6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, and heterocyclyl; and (c) —C(O)Re, —C(O)ORe, —C(O)NRng, —C(NRe)NRng, —ORe, — OC(O)Re, —OC(O)ORe, —OC(O)NRng, —OC(=NRe)NRng, Re, —OS(O)2Re, —OS(O)NRng, —OS(O)2NRng, —NRng, —NReC(O)Rh, —NReC(O)ORf, —NReC(O)NRng, —NReC(=NRh)NRng, O)Rh, —NReS(O)2Rh, —NReS(O)NRng, O)2NRng, —SRe, —S(O)Re, —S(O)2Re, — S(O)NRng, and —S(O)2NRng; wherein each Re, Rf, Rg, and Rh is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl; or (iii) Rf and Rg together with the N atom to which they are attached form heterocyclyl.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the design and production of cells and organisms having biosynthetic tion capabilities for alkenes. The invention, in particular, relates to the design of microbial organism e of producing alkene by introducing one or more nucleic acids encoding an alkene y enzyme.

[0014] In one embodiment, the invention utilizes in silica stoichiometric models of Escherichia cali metabolism that identify metabolic designs for biosynthetic production of alkenes. The results described herein te that metabolic pathways can be designed and recombinantly engineered to achieve the biosynthesis of alkenes in Escherichia cali and other cells or organisms. Biosynthetic production of alkenes, for example, by the in silica designs can be confirmed by construction of strains having the designed lic genotype. These lically engineered cells or organisms also can be subjected to adaptive evolution to further augment alkene biosynthesis, including under conditions ching theoretical maximum growth.

In certain ments, the alkene biosynthesis characteristics of the designed strains make them cally stable and particularly useful in continuous bioprocesses.

Separate strain design strategies were identified with oration of ent non-native or heterologous reaction capabilities into E. cali or other host organisms leading to alkene producing metabolic pathways from alcohols that are produced naturally or that are produced through genetic ering. In silica metabolic designs were identified that resulted in the biosynthesis of alkenes in microorganisms from this substrate or metabolic intermediates.

WO 40383 6 s identified via the computational component of the platform can be put into actual production by genetically engineering any of the predicted metabolic alterations, which lead to the biosynthetic production of s or other intermediate and/or ream products. In yet a further embodiment, strains ting biosynthetic production of these compounds can be further ted to ve evolution to further augment t biosynthesis. The levels of product biosynthesis yield following adaptive evolution also can be predicted by the computational component of the system.

As used herein, the term “non-naturally occurring” when used in reference to a microbial sm or rganism of the invention is intended to mean that the microbial organism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including ype strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other filnctional disruption of the microbial organism’s genetic al.

Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional ations include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary metabolic polypeptides include enzymes or proteins within an alkene biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that is altered from its naturally occurring state. Therefore, non-naturally occurring rganisms can have genetic modifications to nucleic acids encoding metabolic polypeptides, or functional fragments f. Exemplary metabolic ations are disclosed herein.

[0019] As used herein, the term “isolated” when used in reference to a microbial organism is intended to mean an organism that is substantially free of at least one component as the referenced microbial sm is found in nature. The term includes a microbial organism that is removed from some or all components as it is found in its natural environment. The term also includes a ial organism that is removed from some or all components as the microbial organism is found in turally occurring environments. Therefore, an isolated microbial organism is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non- naturally occurring environments. Specific examples of isolated microbial organisms e partially pure microbes, substantially pure microbes and microbes cultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or “microorganism” are intended to mean any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a copic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fiangi. The term also includes cell cultures of any species that can be cultured for the production of a biochemical.

[0021] As used herein, the term “substantially anaerobic” when used in reference to a culture or growth condition is intended to mean that the amount of oxygen is less than about 10% of saturation for dissolved oxygen in liquid media. The term also is intended to e sealed chambers of liquid or solid medium maintained with an here of less than about 1% oxygen.

[0022] “Exogenous” as it is used herein is intended to mean that the nced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding c acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the ng c acid in an expressible form into the microbial sm. When used in reference to a biosynthetic ty, the term refers to an activity that is introduced into the host reference organism. The source can be, for example, a homologous or heterologous ng nucleic acid that expresses the referenced activity following introduction into the host microbial organism. Therefore, the term “endogenous” refers to a referenced molecule or activity that is present in the host. Similarly, the term when used in reference to expression of an encoding nucleic acid refers to expression of an encoding nucleic acid contained within the microbial organism. The term “heterologous” refers to a molecule or activity d from a source other than the referenced species s “homologous” refers to a molecule or activity derived from the host microbial organism. Accordingly, exogenous expression of an ng nucleic acid of the invention can utilize either or both a logous or homologous encoding nucleic acid.

It is understood that when more than one exogenous nucleic acid is included in a microbial organism that the more than one exogenous nucleic acids refers to the referenced ng nucleic acid or biosynthetic activity, as discussed above. It is fiarther understood, as disclosed herein, that such more than one exogenous nucleic acids can be uced into the host ial organism on separate nucleic acid molecules, on polycistronic nucleic acid molecules, or a combination f, and still be considered as more than one exogenous nucleic acid. For example, as disclosed herein a ial organism can be engineered to express two or more ous nucleic acids encoding a desired pathway enzyme or protein. In the case where two exogenous nucleic acids ng a desired activity are introduced into a host microbial sm, it is understood that the two exogenous nucleic acids can be introduced as a single nucleic acid, for example, on a single plasmid, on separate plasmids, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two exogenous nucleic acids. Similarly, it is understood that more than two exogenous nucleic acids can be uced into a host organism in any desired combination, for example, on a single plasmid, on separate ds, can be integrated into the host chromosome at a single site or multiple sites, and still be considered as two or more exogenous nucleic acids, for example three exogenous nucleic acids. Thus, the number of referenced exogenous nucleic acids or biosynthetic activities refers to the number of encoding nucleic acids or the number of biosynthetic activities, not the number of separate nucleic acids introduced into the host organism.

The non-naturally occurring microbal organisms of the invention can contain stable genetic alterations, which refers to microorganisms that can be cultured for greater than five generations without loss of the alteration. lly, stable genetic alterations include modifications that persist r than 10 generations, particularly stable modifications will persist more than about 25 generations, and more ularly, stable genetic modifications will be greater than 50 generations, including indef1nitely.

Those skilled in the art will understand that the genetic alterations, including metabolic modif1cations exemplif1ed herein, are described with reference to a suitable host organism such as E. coli and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway.

However, given the complete genome cing of a wide variety of organisms and the high level of skill in the area of genomics, those d in the art will readily be able to apply the teachings and guidance provided herein to ially all other organisms. For example, the E. 6012' metabolic alterations exemplified herein can readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species. Such genetic alterations e, for example, c alterations of species homologs, in general, and in particular, orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are d by vertical descent and are responsible for substantially the same or identical fianctions in different organisms. For example, mouse epoxide hydrolase and human epoxide hydrolase can be considered orthologs for the biological function of ysis of epoxides. Genes are related by vertical t when, for example, they share sequence similarity of ient amount to te they are homologous, or related by evolution from a common ancestor. Genes can also be considered ogs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the y sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence rity of about 25% to 100% amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less that 25% can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Members of the serine protease family of enzymes, including tissue plasminogen activator and elastase, are considered to have arisen by vertical descent from a common ancestor.

Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product ting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. For the production of a biochemical product, those skilled in the art will understand that the orthologous gene harboring the metabolic activity to be introduced or disrupted is to be chosen for uction of the non-naturally occurring microorganism. An example of ogs ting separable activities is where distinct activities have been separated into distinct gene products between two or more species or within a single species. A specific example is the separation of se proteolysis and plasminogen lysis, two types of serine protease activity, into distinct molecules as plasminogen activator and elastase. A second W0 2013/040383 10 PCT/U82012/055469 example is the separation of mycoplasma 5’-3’ exonuclease and Drosophila DNA polymerase III activity. The DNA polymerase from the first species can be considered an ortholog to either or both of the exonuclease or the polymerase from the second species and vice versa.

In contrast, paralogs are gs related by, for example, duplication followed by evolutionary divergence and have similar or common, but not identical functions. gs can ate or derive from, for example, the same species or from a different species. For example, microsomal epoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II) can be considered paralogs because they represent two distinct enzymes, co-evolved from a common ancestor, that catalyze distinct reactions and have ct functions in the same species. Paralogs are proteins from the same species with cant sequence similarity to each other suggesting that they are homologous, or related through co-evolution from a common ancestor. Groups of paralogous protein families include HipA homologs, luciferase genes, peptidases, and others.

A hologous gene displacement is a nonorthologous gene from one species that can substitute for a nced gene fianction in a different species.

Substitution es, for example, being able to perform substantially the same or a r function in the species of origin ed to the referenced function in the different s. gh generally, a nonorthologous gene displacement will be identifiable as structurally related to a known gene encoding the referenced fianction, less structurally related but functionally r genes and their corresponding gene ts nevertheless will still fall within the meaning of the term as it is used herein. Functional similarity requires, for example, at least some structural similarity in the active site or binding region of a nonorthologous gene product compared to a gene encoding the function sought to be substituted. Therefore, a nonorthologous gene includes, for example, a paralog or an unrelated gene.

Therefore, in identifying and constructing the non-naturally occurring microbial organisms of the invention having alkene biosynthetic capability, those d in the art will understand with applying the teaching and guidance provided herein to a particular species that the identification of metabolic modifications can include identification and inclusion or inactivation of orthologs. To the extent that paralogs and/or nonorthologous gene displacements are t in the referenced microorganism that W0 2013/040383 11 PCT/U82012/055469 encode an enzyme catalyzing a similar or substantially similar metabolic reaction, those skilled in the art also can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can be ined by methods well known to those skilled in the art. For example, inspection of nucleic acid or amino acid sequences for two polypeptides will reveal ce identity and similarities between the compared sequences. Based on such similarities, one skilled in the art can determine if the similarity is sufficiently high to te the proteins are related through evolution from a common ancestor. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal W and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide sequence similarity or identity. ters for sufficient similarity to determine relatedness are computed based on well known s for calculating statistical similarity, or the chance of finding a similar match in a random polypeptide, and the significance of the match determined. A computer comparison of two or more sequences can, if desired, also be optimized visually by those skilled in the art. Related gene products or ns can be expected to have a high similarity, for e, 25% to 100% sequence ty. ns that are unrelated can have an ty which is essentially the same as would be expected to occur by chance, if a database of sufficient size is scanned (about 5%). Sequences between 5% and 24% may or may not represent sufficient homology to conclude that the compared sequences are related. Additional statistical analysis to determine the significance of such matches given the size of the data set can be carried out to determine the relevance of these sequences.

[0032] ary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 5-l999) and the ing parameters: Matrix: 0 BLOSUM62; gap open: 11; gap ion: 1; off: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-l6-l998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off Those skilled in the art will know what modifications can be W0 2013/040383 12 2012/055469 made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

In some embodiments, the invention provides non-naturally occurring ial organisms containing an alkene pathway having at least one exogenous nucleic acid encoding an alkene pathway enzyme expressed in a sufficient amount to convert an alcohol to an alkene as ed in Figure I. In some aspects of the invention, the microbial sm ses an alkene pathway selected from: (I) an alcohol kinase and a ate lyase; (2) a diphosphokinase and a diphosphate lyase; and (3) an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase. In some aspects of the invention, the microbial organism converts an alcohol of Formula (I) R4—c—c—R1 R3 R2 to an alkene of Formula (II) R4 R1 \c——c/ / \ R3 R2 wherein R1, R2, R3, and R4 are each independently (a) hydrogen, cyano, halo, or nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, or cyclyl, each ally substituted with one or more substituents Q; or (c) —C(O)R1a, —C(O)OR1a, —C(O)NR1bR1°, —C(NR1a)NR1bR1°, —0R1a, —OC(O)R1a, —OC(O)OR1a, —OC(O)NR1bR1°, —OC(=NR1a)NR1bR1°, —OS(O)R1a, —OS(O)2R1a, —OS(O)NR1bR1°, 2NRle1°, —NR1bR1°, —NR1aC(O)R1d, —NR1aC(O)ORld, —NR1aC(O)NR1bR1°, —NR1aC(=NR1d)NR1bR1°, —NR1aS(O)R1d, —NR1aS(O)2Rld, —NR1aS(O)NR1bR1°, —NR1aS(O)2NR1bR1°, —SR1a, —S(O)R1a, —S(O)2R1a, —S(O)NR1bR1°, or —S(O)2NR1bR1°; wherein each R”, Rlb, R”, and R1d is independently hydrogen, C1_6 alkyl, C2_6 alkenyl, C2- 6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl; or R1&1 and R10 together with the C and N atoms to which they are attached form heterocyclyl; or R1b and R10 together with the N atom to which they are attached form heterocyclyl; wherein each Q is independently ed from (a) oxo, cyano, halo, and nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, aryl, and heterocyclyl, W0 40383 13 PCT/U82012/055469 each of which is further optionally substituted with one or more, in one embodiment, one, two, three, or four, tuents Qa; and (c) —C(O)Ra, —C(O)ORa, —C(O)NRbR°, — C(NRa)NRbR°, —0Ra, —OC(O)Ra, —OC(O)ORa, —OC(O)NRbR°, —OC(=NRa)NRbR°, —OS(O)Ra, —OS(O)2Ra, —OS(O)NRbR°, —OS(O)2NRbR°, , —NRaC(O)Rd, —NRaC(O)ORd, —NRaC(O)NRbR°, —NRaC(=NRd)NRbR°, —NRaS(O)Rd, —NRaS(O)2Rd, —NRaS(O)NRbR°, —NRaS(O)2NRbR°, —SRa, —S(O)Ra, —S(O)2Ra, RbR°, and —S(O)2NRbR°, wherein each Ra, Rb, R°, and Rd is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 l, C3_7 cycloalkyl, C644 aryl, C745 l, heteroaryl, or heterocyclyl, each optionally tuted with one or more, in one embodiment, one, two, three, or four, tuents Qa; or (iii) Rb and Rc together with the N atom to which they are attached form heterocyclyl, optionally substituted with one or more, in one ment, one, two, three, or four, substituents Qa; wherein each Qa is independently selected from the group consisting of (a) oxo, cyano, halo, and nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, aryl, and heterocyclyl; and (c) —C(O)Re, —C(O)ORe, —C(O)NRng, — C(NRe)NRng, —0Re, —OC(O)Re, —OC(O)ORe, —OC(O)NRng, —OC(=NRe)NRng, Re, —OS(O)2Re, —OS(O)NRng, —OS(O)2NRng, —NRng, —NReC(O)Rh, —NReC(O)ORf, —NReC(O)NRng, —NReC(=NRh)NRng, —NReS(O)Rh, —NReS(O)2Rh, —NReS(O)NRng, O)2NRng, — SR3, —S(O)Re, —S(O)2Re, —S(O)NRng, and —S(O)2NRng; wherein each Re, Rf, Rg, and Rh is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C6- 14 aryl, C7_1 5 aralkyl, heteroaryl, or heterocyclyl; or (iii) Rf and Rg together with the N atom to which they are attached form heterocyclyl. It is also understood that R1, R2, R3, and R4 are each ndently same between the alcohol and the alkene. In otherwords, the R1 of the alcohol is the same as the R1 of the alkene, the R2 of the alcohol is the same as the R2 of the alkene, the R3 of the alcohol is the same as the R3 of the alkene and the R4 of the alcohol is the same as the R4 of the alkene.

The term “alkyl” refers to a linear or branched saturated monovalent hydrocarbon radical, wherein the alkyl may optionally be substituted with one or more substituents Q as described herein. For example, C1_6 alkyl refers to a linear saturated monovalent hydrocarbon radical of l to 6 carbon atoms or a branched saturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkyl is a linear saturated monovalent hydrocarbon radical that has 1 to 20 (C1_20), l to 15 (C1- ), l to 10 (C140), or 1 to 6 (C1_6) carbon atoms, or ed saturated monovalent W0 2013/040383 14 PCT/U82012/055469 arbon l of 3 to 20 (€3-20), 3 to 15 (C345), 3 to 10 (€3-10), or 3 to 6 (C3_6) carbon atoms. As used , linear C1_6 and branched C3_6 alkyl groups are also ed as “lower alkyl.” Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including all isomeric forms), n-propyl, isopropyl, butyl (including all isomeric forms), l, isobutyl, tyl, t—butyl, pentyl ding all isomeric forms), and hexyl (including all isomeric forms).

The term “alkenyl” refers to a linear or branched monovalent hydrocarbon radical, which contains one or more, in one embodiment, one to five, in another ment, one, carbon-carbon double bond(s). The alkenyl may be optionally substituted with one or more substituents Q as described herein. The term yl” embraces radicals having a “cis” or “trans” configuration or a mixture thereof, or alternatively, a “Z” or “E” configuration or a mixture thereof, as appreciated by those of ordinary skill in the art. For example, C2_6 alkenyl refers to a linear unsaturated monovalent arbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkenyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2_20), 2 to 15 (C245), 2 to (C240), or 2 to 6 (C2_6) carbon atoms, or a branched monovalent hydrocarbon radical of 3 to 20 (€3-20), 3 to 15 (C345), 3 to 10 (€3-10), or 3 to 6 (C3_6) carbon atoms. Examples of alkenyl groups include, but are not d to, ethenyl, propen-l-yl, propenyl, allyl, butenyl, and ylbutenyl.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbon radical, which contains one or more, in one ment, one to five, in another embodiment, one, carbon-carbon triple bond(s). The alkynyl may be optionally substituted with one or more substituents Q as described herein. For example, C2_6 alkynyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms. In certain embodiments, the alkynyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2_20), 2 to 15 (C245), 2 to 10 , or 2 to 6 (C2_6) carbon atoms, or a branched monovalent arbon radical of 3 to 20 (€3-20), 3 to 15 (C345), 3 to 10 (€3-10), or 3 to 6 (C3_6) carbon atoms. es of alkynyl groups include, but are not limited to, ethynyl (—CECH), propynyl (including all isomeric forms, e.g., l-propynyl (—CECCHg) and propargyl (— CHZCECH», butynyl (including all isomeric forms, e.g., l-butyn-l-yl and 2-butyn-l-yl), 15 2012/055469 pentynyl (including all ic forms, 6.g. , l -pentyn- l -yl and l -methylbutyn- l -yl), and hexynyl (including all isomeric forms, 6.g. , l-hexyn-l-yl).

The term alkyl” refers to a cyclic monovalent hydrocarbon radical, which may be optionally substituted with one or more substituents Q as described herein.

In one embodiment, lkyl groups may be saturated or unsaturated but non-aromatic, and/or bridged, and/or non-bridged, and/or fused bicyclic groups. In certain embodiments, the cycloalkyl has from 3 to 20 (€3-20), from 3 to 15 (€3-15), from 3 to 10 (€3-10), or from 3 to 7 (C3-7) carbon atoms. Examples of cycloalkyl groups include, but are not limited to, ropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptenyl, bicyclo [2. l . l ]hexyl, bicyclo[2.2. l]heptyl, decalinyl, and tyl.

The term “aryl” refers to a monovalent monocyclic aromatic group and/or monovalent polycyclic aromatic group that contain at least one aromatic carbon ring. In certain embodiments, the aryl has from 6 to 20 (C6_20), from 6 to 15 , or from 6 to 10 (C640) ring atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. Aryl also refers to bicyclic or tricyclic carbon rings, where one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl). In certain embodiments, aryl may be ally substituted with one or more substituents Q as described herein.

The term “aralkyl” or “arylalkyl” refers to a monovalent alkyl group substituted with one or more aryl groups. In n embodiments, the aralkyl has from 7 to 30 (C7_30), from 7 to 20 ), or from 7 to 16 (€7-16) carbon atoms. Examples of aralkyl groups include, but are not limited to, benzyl, 2-phenylethyl, and 3-phenylpropyl.

In certain embodiments, aralkyl are optionally substituted with one or more substituents Q as described herein.

The term “heteroaryl” refers to a monovalent clic aromatic group or lent polycyclic aromatic group that contain at least one aromatic ring, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N in the ring. Heteroaryl groups are bonded to the rest of a molecule through the aromatic ring. Each ring of a heteroaryl group can contain one or two 0 atoms, one or two W0 2013/040383 16 S atoms, and/or one to four N atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms.

Examples of monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl, lyl, thienyl, tetrazolyl, triazinyl, and triazolyl. Examples of bicyclic aryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, hiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, opyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofilranyl, isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Examples of tricyclic aryl groups e, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, inyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain ments, heteroaryl may also be optionally substituted with one or more substituents Q as described herein.

The term “heterocyclyl” or “heterocyclic” refers to a lent monocyclic non-aromatic ring system or lent polycyclic ring system that contains at least one non-aromatic ring, n one or more of the non-aromatic ring atoms are heteroatoms independently selected from O, S, and N; and the ing ring atoms are carbon atoms.

In certain embodiments, the heterocyclyl or heterocyclic group has from 3 to 20, from 3 to , from 3 to 10, from 3 to 8, from 4 to 7, or from 5 to 6 ring atoms. Heterocyclyl groups are bonded to the rest of a molecule through the non-aromatic ring. In certain embodiments, the heterocyclyl is a monocyclic, bicyclic, lic, or tetracyclic ring system, which may be fused or bridged, and in which nitrogen or sulfur atoms may be optionally oxidized, nitrogen atoms may be optionally quatemized, and some rings may be partially or fillly saturated, or aromatic. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom which results in the on of a stable compound. Examples of such heterocyclic groups include, but are not limited to, azepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl, benzopyranyl, benzotetrahydrofiaranyl, benzotetrahydrothienyl, benzothiopyranyl, benzoxazinyl, B- inyl, chromanyl, chromonyl, inyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, l,4-dithianyl, furanonyl, imidazolidinyl, olinyl, indolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl, isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, yl, piperazinyl, dinyl, 4-piperidonyl, pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, lidinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl, and 1,3,5- anyl. In certain ments, heterocyclic may also be optionally substituted with one or more substituents Q as described .

The term “halogen”, “halide” or “halo” refers to fluorine, chlorine, e, and/or iodine.

The term “optionally substituted” is intended to mean that a group or substituent, such as an alkyl, alkylene, heteroalkylene, alkenyl, alkenylene, heteroalkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, aryl, arylene, aralkyl, aryl, heteroarylene, heterocyclyl, or heterocyclylene group, may be substituted with one or more substituents Q, each of which is independently selected from, e.g., (a) C1_6 alkyl, C2_6 l, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 l, heteroaryl, and heterocyclyl, each of which is filrther optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; and (b) oxo (=0), halo, cyano (— CN), nitro (—N02), —C(O)Ra, —C(O)ORa, RbR°, —C(NRa)NRbR°, —0Ra, —OC(O)Ra, —OC(O)ORa, —OC(O)NRbR°, —OC(=NRa)NRbR°, —OS(O)Ra, —OS(O)2Ra, —OS(O)NRbR°, — OS(O)2NRbR°, —NRbR°, —NRaC(O)Rd, —NRaC(O)ORd, —NRaC(O)NRbR°, — NRaC(=NRd)NRbR°, —NRaS(O)Rd, —NRaS(O)2Rd, —NRaS(O)NRbR°, —NRaS(O)2NRbR°, — SRa, —S(O)Ra, —S(O)2Ra, —S(O)NRbR°, and —S(O)2NRbR°, wherein each Ra, Rb, R°, and Rd is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C6_ 14 aryl, C745 aralkyl, heteroaryl, or cyclyl, each optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; or (iii) Rb and Rc together with the N atom to which they are attached form heteroaryl or heterocyclyl, optionally substituted with one or more, in one ment, one, two, three, or four, substituents Qa. As used herein, all groups that can be substituted are “optionally substituted,” unless otherwise specified.

W0 2013/040383 18 PCT/U82012/055469 In one embodiment, each Qa is independently selected from the group consisting of (a) oxo, cyano, halo, and nitro; and (b) C1_6 alkyl, C2_6 l, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, and heterocyclyl; and (c) —C(O)Re, — C(O)ORe, —C(O)NRng, —C(NRe)NRng, —0Re, —OC(O)Re, —OC(O)ORe, NRng, — OC(=NRe)NRng, —OS(O)Re, 2Re, —OS(O)NRng, —OS(O)2NRng, —NRng, — NReC(O)Rh, —NReC(O)ORh, —NReC(O)NRng, —NReC(=NRh)NRng, —NReS(O)Rh, — NReS(O)2Rh, —NReS(O)NRng, —NReS(O)2NRng, —SRe, e, —S(O)2Re, —S(O)NRng, and —S(O)2NRng; wherein each Re, Rf, Rg, and Rh is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl; or (iii) Rf and Rg together with the N atom to which they are attached form heteroaryl or heterocyclyl.

Figure 1 shows pathways for converting an alcohol to an alkene via a phosphate or diphosphate intermediate. In step A, an alcohol is activated to an alkyl phosphate by a kinase. The alkyl phosphate is then fiarther activated to an alkyl phate (Step D) or converted to an alkene by a phosphate lyase or alkene synthase (step B). Altemately, the alcohol is directly converted to the alkyl diphosphate intermediate by a diphosphokinse (step C). The release of diphosphate from alkyl diphosphate by an alkene synthase or phate lyase yields an alkene. Exemplary alcohol sors and alkene ts are listed in the table below.

Alcohol Alkene Ethanol Eth lene n-Pro o anol Pro 0 lene Iso o ro o anol Pro 0 lene n-Butanol But-l-ene Isobutanol Isobu lene Tert-butanol Isobut lene Butanol Butene or butene Pentanol Pent- l -ene 3-meth lbutanol 3-meth lbut- l -ene Pentanol Pentene or o entene Pentan-3 -ol Pentene 2-Meth lbutanol 2-meth lbut- l -ene 3-Meth lbutanol 3-Meth lbut- l -ene 2-Methylbutanol 2-Methylbut- l -ene or 2-Meth lbutene 3-Meth lbut-3 -enol Iso o rene 2-Meth lbut-3 -enol ISO 0 rene W0 2013/040383 19 2012/055469 Alcohol Alkene 2-Meth lbutenol 3-Meth lbuta-l,2-diene 2-Meth lbut—3-en-l-ol Iso-rene 3-Meth lbutenol Is0orene Butenol 1 ,3 -Butadiene Butenol l ,3-Butadiene l-Phen lethanol St rene 2-Phen lethanol St rene Dimeth lall lalcohol Is0orene Butenol 1 ,3 -Butadiene Accordingly, in some aspects, the invention provides a non-naturally occurring microbial organism ning an alkene pathway having at least one exogenous nucleic acid encoding an alkene y enzyme expressed in a sufficient amount to convert an alcohol to an alkene as depicted in the table above. In some aspects of the invention, the microbial organism comprises an alkene y selected from: (1) an alcohol kinase and a phosphate lyase; (2) a diphosphokinase and a diphosphate lyase; and (3) an alcohol kinase, an alkyl phosphate kinase and a phate lyase. In some aspects, the ial ism of the ion converts ethanol to ethylene, n-propanol t0 propylene, isopropanol to propylene, n-butanol t0 but-l-ene, isobutanol t0 isobutylene, tert-butanol to isobutylene, butanol to but-l-ene or butene, pentan-l-ol to pent-l-ene, 3- methylbutan-l-ol t0 3-methylbut-l-ene, ol t0 pentene, pentalol t0 pent ene, 2-methylbutanol t0 2-methylbut- l -ene, 3 -methylbutanol t0 3 -methylbut- l -ene, 2-methylbutanol to 2-methylbut- l -ene or 2-methylbutene, 3-methylbuten-l -ol to isoprene, 2-methylbutenol t0 isoprene, 2-methylbutenol to 3-methylbuta-l,2- diene, 2-methylbut-3 -en01 to isoprene, 3 -methylbut-3 -enol to isoprene, but-3 -en01 to l,3-butadiene, butenol t0 l,3-butadiene, ylethanol t0 styrene, 2- phenylethanol t0 styrene, dimethylallyl alcohol to isoprene, or buten-l-ol to 1,3- butadiene.

[0047] In some embodiments of the invention, the non-naturally occurring microbial organism comprises two or three exogenous nucleic acids each encoding an alkene y enzyme. For example, two exogenous nucleic acids can encode an alcohol kinase and a phosphate lyase, or alternatively a diphosphokinase and a diphosphate lyase. In some aspects of the invention, non-naturally occurring microbial sm can include three exogenous nucleic acids encoding an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase. The invention also provides that the at least one exogenous nucleic W0 2013/040383 20 PCT/U82012/055469 acid can be a heterologous nucleic acid. The invention still fiarther provides that the non- naturally occurring microbial organism can be in a substantially anaerobic culture medium.

In an additional embodiment, the invention es a non-naturally occurring microbial organism having an alkene pathway, wherein the non-naturally occurring microbial organism comprises at least one ous nucleic acid encoding an enzyme or protein that ts a ate to a product selected from the group consisting of an alcohol to an alkyl phosphate, an alcohol to an alkyl phate, an alkyl phosphate to an alkyl diphosphate, an alkyl phosphate to an alkene or an alkyl diphosphate to an .

One skilled in the art will understand that these are merely exemplary and that any of the substrate-product pairs disclosed herein suitable to produce a desired product and for which an appropriate activity is available for the conversion of the substrate to the product can be readily determined by one skilled in the art based on the teachings . Thus, the invention provides a non-naturally occurring ial organism ning at least one ous nucleic acid encoding an enzyme or protein, where the enzyme or protein converts the substrates and products of an alkene pathway, such as that shown in Figure l.

While generally described herein as a microbial sm that contains an alkene pathway, it is tood that the invention additionally provides a non-naturally occurring microbial organism comprising at least one exogenous nucleic acid encoding an alkene y enzyme expressed in a sufficient amount to produce an intermediate of an alkene pathway. For e, as disclosed herein, an alkene pathway is exemplified in Figure 1. Therefore, in addition to a microbial organism containing an alkene pathway that produces alkene, the invention additionally provides a non-naturally occurring microbial organism sing at least one exogenous nucleic acid encoding an alkene pathway enzyme, where the microbial organism produces an alkene pathway ediate, for example, an alkyl phosphate or an alkyl diphosphate.

It is understood that any of the pathways disclosed herein, as described in the Examples and exemplified in the Figures, including the pathways of Figure 1, can be utilized to generate a non-naturally occurring microbial organism that produces any pathway intermediate or product, as desired. As disclosed herein, such a microbial organism that produces an intermediate can be used in combination with another microbial organism expressing downstream pathway enzymes to e a desired product.

However, it is understood that a turally occurring microbial organism that produces W0 2013/040383 21 an alkene pathway ediate can be utilized to produce the intermediate as a desired product.

The invention is described herein with general reference to the metabolic reaction, nt or product thereof, or with specific reference to one or more nucleic acids or genes encoding an enzyme associated with or zing, or a protein associated with, the referenced metabolic reaction, reactant or product. Unless otherwise expressly stated herein, those skilled in the art will understand that reference to a reaction also constitutes reference to the reactants and products of the reaction. Similarly, unless otherwise expressly stated herein, nce to a reactant or product also references the reaction, and reference to any of these metabolic constituents also references the gene or genes encoding the enzymes that catalyze or ns involved in the referenced reaction, reactant or product. Likewise, given the well known fields of metabolic biochemistry, enzymology and genomics, reference herein to a gene or encoding nucleic acid also constitutes a reference to the corresponding d enzyme and the reaction it catalyzes or a protein associated with the reaction as well as the nts and products of the reaction.

The non-naturally ing microbial sms of the invention can be produced by introducing expressible nucleic acids encoding one or more of the enzymes or proteins ipating in one or more alkene thetic pathways. Depending on the host microbial organism chosen for biosynthesis, nucleic acids for some or all of a particular alkene biosynthetic pathway can be expressed. For example, if a chosen host is nt in one or more enzymes or proteins for a d biosynthetic pathway, then expressible nucleic acids for the deficient enzyme(s) or protein(s) are introduced into the host for subsequent exogenous sion. Alternatively, if the chosen host exhibits endogenous expression of some pathway genes, but is deficient in others, then an encoding nucleic acid is needed for the nt enzyme(s) or protein(s) to achieve alkene thesis. Thus, a non-naturally occurring microbial organism of the invention can be produced by introducing exogenous enzyme or protein activities to obtain a desired biosynthetic pathway or a desired biosynthetic pathway can be obtained by introducing one or more exogenous enzyme or protein activities that, together with one or more endogenous enzymes or proteins, produces a desired t such as alkene.

Host microbial organisms can be selected from, and the non-naturally occurring microbial organisms generated in, for example, bacteria, yeast, fiangus or any of W0 2013/040383 22 PCT/U82012/055469 a variety of other microorganisms applicable to tation processes. Exemplary bacteria include species selected from Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum iciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus , Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, and Pseudomonas . Exemplary yeasts or fiangi include species selected from Saccharomyces siae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris, Rhizopus us, Rhizobus oryzae, Yarrowia lipolytica, and the like. E. coli is a ularly useful host organism since it is a well characterized microbial organism suitable for c engineering. Other particularly useful host organisms include yeast such as Saccharomyces cerevisiae. It is understood that any suitable microbial host organism can be used to uce metabolic and/or genetic modifications to produce a desired product.

Depending on the alkene biosynthetic pathway constituents of a ed host microbial organism, the non-naturally occurring microbial organisms of the invention will include at least one ously expressed alkene pathway-encoding nucleic acid and up to all encoding nucleic acids for one or more alkene biosynthetic pathways. For example, alkene biosynthesis can be established in a host deficient in a pathway enzyme or protein through exogenous expression of the corresponding encoding nucleic acid. In a host deficient in all enzymes or proteins of an alkene pathway, exogenous expression of all enzyme or proteins in the pathway can be included, although it is understood that all enzymes or proteins of a y can be expressed even if the host ns at least one of the pathway enzymes or proteins. For example, exogenous expression of all enzymes or proteins in a pathway for production of alkene can be included, such as an alcohol kinase and a phosphate lyase, or alternatively a diphosephokinase and a diphosphate lyase, or alternatively an alcohol , an alkyl phosphate kinase and a diphosphate lyase.

Given the teachings and guidance provided herein, those skilled in the art will understand that the number of encoding c acids to introduce in an expressible form will, at least, parallel the alkene pathway deficiencies of the selected host microbial sm. Therefore, a non-naturally occurring ial organism of the invention can have one, two or three up to all nucleic acids encoding the enzymes or proteins W0 2013/040383 23 PCT/U82012/055469 constituting an alkene biosynthetic pathway disclosed herein. In some embodiments, the non-naturally occurring microbial organisms also can include other genetic modifications that facilitate or optimize alkene biosynthesis or that confer other useful fianctions onto the host microbial organism. One such other functionality can include, for example, augmentation of the synthesis of one or more of the alkene pathway precursors such as an alcohol disclosed herein.

Generally, a host microbial organism is selected such that it produces the precursor of an alkene y, either as a naturally produced molecule or as an engineered product that either provides de novo tion of a desired precursor or increased production of a precursor naturally produced by the host microbial sm.

For example, ethanol is produced naturally in a host organism such as E. 0011'. A host organism can be engineered to increase production of a sor, as disclosed herein. In addition, a microbial sm that has been engineered to produce a d precursor can be used as a host organism and further engineered to express enzymes or proteins of an alkene pathway.

In some embodiments, a non-naturally occurring microbial organism of the invention is generated from a host that contains the enzymatic capability to synthesize alkene. In this specific embodiment it can be useful to increase the sis or accumulation of an alkene pathway t to, for example, drive alkene pathway reactions toward alkene production. Increased synthesis or accumulation can be accomplished by, for example, overexpression of nucleic acids ng one or more of the described alkene pathway enzymes or proteins. Overexpression of the enzyme or enzymes and/or n or proteins of the alkene pathway can occur, for example, through exogenous expression of the endogenous gene or genes, or through ous expression of the heterologous gene or genes. Therefore, naturally occurring organisms can be readily generated to be non-naturally occurring microbial sms of the invention, for example, producing alkene, through pression of one, two, or three, that is, up to all nucleic acids encoding alkene biosynthetic pathway enzymes or proteins.

In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme in the alkene biosynthetic pathway.

In particularly useful ments, exogenous expression of the encoding nucleic acids is employed. Exogenous sion confers the ability to custom tailor the W0 2013/040383 24 PCT/U82012/055469 expression and/or tory elements to the host and application to achieve a desired expression level that is controlled by the user. However, endogenous expression also can be utilized in other embodiments such as by ng a negative tory or or induction of the gene’s promoter when linked to an inducible promoter or other regulatory element. Thus, an endogenous gene having a naturally occurring ble promoter can be up-regulated by providing the appropriate inducing agent, or the regulatory region of an endogenous gene can be engineered to incorporate an inducible regulatory element, thereby allowing the regulation of increased expression of an endogenous gene at a desired time. Similarly, an inducible promoter can be included as a regulatory element for an exogenous gene introduced into a non-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one or more exogenous c acids can be introduced into a microbial organism to produce a non- naturally occurring ial organism of the invention. The nucleic acids can be introduced so as to confer, for example, an alkene biosynthetic y onto the microbial organism. Alternatively, encoding nucleic acids can be introduced to produce an intermediate microbial organism having the thetic capability to catalyze some of the required reactions to confer alkene biosynthetic capability. For example, a non-naturally occurring ial organism having an alkene biosynthetic pathway can comprise at least two exogenous nucleic acids encoding desired enzymes or proteins, such as the combination of an alcohol kinase and a phosphate lyase, or alternatively a diphosphokinase and a diphosphate lyase, and the like. Thus, it is understood that any combination of two or more enzymes or proteins of a biosynthetic pathway can be included in a turally ing microbial organism of the invention. Similarly, it is understood that any combination of three or more enzymes or proteins of a thetic pathway can be included in a non-naturally occurring microbial organism of the invention, for e, an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase, and so forth, as desired, so long as the combination of enzymes and/or proteins of the desired biosynthetic pathway results in tion of the corresponding desired product.

In addition to the biosynthesis of alkene as described herein, the non-naturally occurring microbial sms and methods of the invention also can be ed in various combinations with each other and with other microbial organisms and methods well known in the art to achieve product biosynthesis by other routes. For example, one alternative to produce alkene other than use of the alkene producers is through addition of another microbial organism e of converting an alkene pathway intermediate to alkene. One such procedure includes, for example, the tation of a microbial organism that produces an alkene pathway intermediate. The alkene pathway intermediate can then be used as a substrate for a second microbial organism that converts the alkene pathway intermediate to alkene. The alkene pathway intermediate can be added directly to another culture of the second organism or the original culture of the alkene pathway intermediate producers can be depleted of these microbial sms by, for example, cell separation, and then subsequent addition of the second organism to the fermentation broth can be utilized to produce the final product without intermediate purification steps.

[0061] In other embodiments, the non-naturally occurring microbial organisms and methods of the invention can be assembled in a wide variety of subpathways to achieve biosynthesis of, for e, . In these embodiments, biosynthetic pathways for a desired product of the invention can be segregated into different microbial organisms, and the different microbial organisms can be co-cultured to produce the final product. In such a biosynthetic scheme, the t of one microbial organism is the substrate for a second microbial organism until the final product is synthesized. For example, the biosynthesis of alkene can be accomplished by constructing a microbial organism that contains biosynthetic pathways for conversion of one pathway intermediate to another pathway intermediate or the product. Alternatively, alkene also can be biosynthetically ed from microbial organisms through co-culture or co-fermentation using two organisms in the same , where the first microbial organism produces an alkyl phosphate or alkyl diphosphate intermediate and the second ial organism converts the intermediate to alkene.

Given the teachings and guidance provided herein, those skilled in the art will understand that a wide variety of combinations and permutations exist for the non- naturally occurring microbial organisms and s of the invention together with other ial organisms, with the ture of other non-naturally occurring microbial organisms haVing subpathways and with combinations of other chemical and/or biochemical procedures well known in the art to produce alkene.

[0063] Sources of encoding c acids for an alkene pathway enzyme or protein can include, for example, any species where the encoded gene t is capable of catalyzing the referenced reaction. Such species include both yotic and eukaryotic sms including, but not limited to, ia, including archaea and eubacteria, and eukaryotes, W0 2013/040383 26 PCT/U82012/055469 including yeast, plant, insect, animal, and mammal, including human. Exemplary species for such sources include, for example, Escherichia coli, Abies grandis, Acetobacter pasteurians, Acinetobacter sp. strain M-1, Arabidopsis thaliana, Arabidopsis thaliana col, Aspergillus terreus N1H2624, Bacillus amyloliquefaciens Bacillus cereus, Bos Taurus, Bradyrhizobiumjaponicum USDA110, Burkholderia um, Burkholderia xenovorans, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium beijerinckii NRRL B593, Clostridium num, Clostridium kluyveri DSM 555, Clostridium saccharoperbutylacetonicum, Comamonas sp. CNB—I, s sativus, Cupriavidus taiwanensis, Enterococcusfaecalis, Escherichia coli C, ichia coli K12, lO Escherichia coli W, Geobacillus thermoglucosidasius Homo sapiens, Klebsiella pneumonia, romyces lactis, Lactococcus lactis, Malus x domestica, Mesorhizobium loti, Methanocaldococcusjannaschii, Methanosarcina mazei, Mycobacterium tuberculosis, Mycoplasma pneumoniae M129, Neurospora crassa, Oryctolagus cuniculus, Picea abies, Populus alba, Populus tremula x Populus alba, Pseudomonas nosa, Pseudomonas putida, Pseudomonas sp. CF600, Pueraria Montana, Pyrococcusfuriosus, Ralstonia eutropha, Ralstonia eutropha H16, nia metallidurans, Rattus norvegicus, Rhodococcus ruber, Saccharomyces cerevisiae, ella enteric, Solanum lycopersicum, Staphylococcus aureus, ococcus pneumonia, omyces sp. ACT-1, Thermoanaerobacter brockii HTD4, toga maritime MSB8, Thermus thermophilus, Zea mays, ea ramigera, Zymomonas mobilis, as well as other exemplary species disclosed herein or available as source organisms for corresponding genes. r, with the complete genome sequence available for now more than 550 species (with more than half of these available on public databases such as the NCBI), including 395 microorganism genomes and a variety of yeast, fungi, plant, and mammalian genomes, the identification of genes encoding the requisite alkene biosynthetic ty for one or more genes in related or distant species, including for example, homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations n organisms is routine and well known in the art. ingly, the metabolic alterations allowing biosynthesis of alkene described herein with reference to a particular organism such as E. coli can be readily applied to other microorganisms, including prokaryotic and eukaryotic organisms alike. Given the teachings and guidance provided herein, those d in the art will know that a lic alteration exemplified in one organism can be applied equally to other organisms.

WO 40383 27 In some instances, such as when an alternative alkene biosynthetic pathway exists in an unrelated species, alkene biosynthesis can be conferred onto the host species by, for example, ous expression of a paralog or paralogs from the unrelated species that catalyzes a r, yet non-identical metabolic reaction to replace the referenced reaction. Because certain differences among lic networks exist between different sms, those skilled in the art will understand that the actual gene usage between ent sms may differ. However, given the teachings and guidance provided herein, those skilled in the art also will understand that the ngs and methods of the ion can be applied to all microbial organisms using the cognate metabolic tions to those exemplified herein to construct a ial organism in a s of interest that will synthesize alkene.

Methods for constructing and testing the expression levels of a non-naturally occurring alkene-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999).

Exogenous nucleic acid sequences involved in a pathway for production of alkene can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, uction, ection, and ultrasound transformation. For exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N—terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. For example, removal of a ondrial leader sequence led to increased expression in E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)). For ous expression in yeast or other otic cells, genes can be expressed in the cytosol without the addition of leader sequence, or can be targeted to mitochondrion or other organelles, or targeted for secretion, by the addition of a suitable targeting sequence such as a mitochondrial targeting or secretion signal suitable for the host cells. Thus, it is understood that appropriate modifications to a nucleic acid sequence to remove or include a targeting sequence can be incorporated into an exogenous nucleic acid sequence to impart desirable properties. Furthermore, genes can be subjected W0 2013/040383 28 PCT/U82012/055469 to codon optimization with techniques well known in the art to achieve zed expression of the proteins.

An expression vector or s can be constructed to include one or more alkene biosynthetic pathway encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, Viral vectors, es and artificial chromosomes, including vectors and selection sequences or markers le for stable integration into a host chromosome. Additionally, the expression vectors can include one or more selectable marker genes and appropriate expression control ces. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply al nts not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous ng nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in te expression vectors. For single vector expression, the encoding c acids can be operationally linked to one common expression control ce or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using s well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification ofmRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is filrther tood that expression levels can be optimized to obtain sufficient sion using methods well known in the art and as sed herein.

The invention additionally provides methods of using the ial organisms disclosed herein to produce an alkene, by culturing a non-naturally occurring microbial organism containing an alkene pathway as described herein under conditions and for a sufficient period of time to produce an alkene. In some s of the method, the W0 2013/040383 29 PCT/U82012/055469 microbial organism used in the method can produce an alkene, wherein the alkene is a compound of Formula (11) R4 R1 \C=C/ R3/ \R2 wherein R1, R2, R3, and R4 are each independently (a) hydrogen, cyano, halo, or nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, or heterocyclyl, each optionally substituted with one or more substituents Q; or (c) —C(O)R1a, —C(O)OR1a, —C(O)NR1bR1°, —C(NR1a)NR1bR1°, —0R1a, —OC(O)R1a, — R1a, —OC(O)NR1bR1°, —OC(=NR1a)NR1bR1°, —OS(O)R1a, —OS(O)2R1a, —OS(O)NR1bR1°, —OS(O)2NRle1°, —NR1bR1°, —NR1aC(O)R1d, —NR1aC(O)ORld, (O)NR1bR1°, (=NR1d)NR1bR1°, —NR1aS(O)R1d, —NR1aS(O)2Rld, —NR1aS(O)NR1bR1°, —NR1aS(O)2NR1bR1°, —SR1a, —S(O)R1a, —S(O)2R1a, —S(O)NR1bR1°, or —S(O)2NR1bR1°; wherein each R”, Rlb, R”, and R1d is independently hydrogen, C1_6 alkyl, C2_6 alkenyl, C2- 6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, or heterocyclyl; or R1&1 and R10 together with the C and N atoms to which they are attached form heterocyclyl; or R1b and R10 together with the N atom to which they are attached form cyclyl; wherein each Q is independently ed from (a) oxo, cyano, halo, and nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, aryl, and heterocyclyl, each of which is r optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; and (c) —C(O)Ra, Ra, —C(O)NRbR°, — NRbR°, —0Ra, —OC(O)Ra, —OC(O)ORa, —OC(O)NRbR°, —OC(=NRa)NRbR°, —OS(O)Ra, —OS(O)2Ra, NRbR°, —OS(O)2NRbR°, —NRbR°, —NRaC(O)Rd, —NRaC(O)ORd, —NRaC(O)NRbR°, —NRaC(=NRd)NRbR°, —NRaS(O)Rd, —NRaS(O)2Rd, —NRaS(O)NRbR°, —NRaS(O)2NRbR°, —SRa, —S(O)Ra, —S(O)2Ra, —S(O)NRbR°, and —S(O)2NRbR°, wherein each Ra, Rb, RC, and Rd is independently (i) hydrogen; (ii) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, or heterocyclyl, each optionally substituted with one or more, in one embodiment, one, two, three, or four, substituents Qa; or (iii) Rb and Rc together with the N atom to which they are attached form heterocyclyl, optionally substituted with one or more, in one embodiment, one, two, W0 2013/040383 30 PCT/U82012/055469 three, or four, substituents Qa; wherein each Qa is independently selected from the group consisting of (a) oxo, cyano, halo, and nitro; (b) C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C7_1 5 aralkyl, heteroaryl, and heterocyclyl; and (c) —C(O)Re, — C(O)ORe, —C(O)NRng, —C(NRe)NRng, —0Re, —OC(O)Re, —OC(O)ORe, —OC(O)NRng, — OC(=NRe)NRng, —OS(O)Re, —OS(O)2Re, NRng, —OS(O)2NRng, —NRng, —NReC(O)Rh, —NReC(O)ORf, —NReC(O)NRng, —NReC(=NRh)NRng, —NReS(O)Rh, —NReS(O)2Rh, —NReS(O)NRng, O)2NRng, — SR3, —S(O)Re, —S(O)2Re, —S(O)NRng, and —S(O)2NRng; wherein each R3, Rf, Rg, and Rh is ndently (i) hydrogen; (ii) C1_6 alkyl, C2_6 l, C2_6 alkynyl, C3_7 cycloalkyl, C644 aryl, C745 aralkyl, heteroaryl, or heterocyclyl; or (iii) Rf and Rg together with the N atom to which they are attached form heterocyclyl.

In some aspects, the microbial organism used in the method disclosed herein can produce an alkene, wherein the alkene is a compound selected from, but are not limited to, Ethylene, Propylene, Propylene, But-l-ene, Isobutylene, Isobutylene, But-l- ene, butene, Pent-l-ene, 3-methylbut-l-ene, Pentene, ylbut-l-ene, 3- Methylbut- l -ene, 2-Methylbut- l -ene, ylbutene, Isoprene, 3-Methylbuta-l ,2- diene, l,3-Butadiene and Styrene.

In some embodiments the alkene product is gaseous and has limited solubility in the culture broth under the conditions of the process. This is advantageous, as removal of the gas from the on vessel can drive the alkene-forming pathway reactions in the forward ion.

Elevated ature can fiarther limit solubility of the alkene products. A desirable property of the microorganism containing the alkene-producing pathway is the ability to grow at elevated temperatures. ary thermophilic and heat-tolerant organisms include Thermus aquaticus, bacteria of the genus Clostrz'dz'um and rganisms of the genera Thermotoga and Aquz’fex. A desired property of the alkene- producing pathway enzymes is the ability to catalyze the desired reactions at elevated temperatures. Such enzymes can be isolated from thermophilic organisms or can be obtained by nizing available enzymes and screening or ing for increased ty under increased temperature conditions.

W0 2013/040383 31 PCT/U82012/055469 Suitable purification and/or assays to test for the production of an alkene can be performed using well known methods. Suitable replicates such as triplicate cultures can be grown for each ered strain to be tested. For example, product and byproduct formation in the ered production host can be monitored. The final product and intermediates, and other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS (Liquid tography-Mass Spectroscopy) or other le ical s using routine procedures well known in the art. The release of product in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual glucose can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol.

Bioeng. 90:775-779 (2005)), or other suitable assay and detection methods well known in the art. The individual enzyme or protein activities from the exogenous DNA sequences can also be assayed using methods well known in the art. Gaseous s can be analyzed by gas chromatography (GC) coupled with a flame ionization detector, and r by GC-MS.

The alkene can be separated from other ents in the culture using a variety of methods well known in the art. Such separation methods include, for example, extraction procedures as well as methods that include continuous liquid-liquid extraction, pervaporation, membrane filtration, membrane tion, reverse s, electrodialysis, distillation, crystallization, centrifugation, extractive ion, ion exchange chromatography, size exclusion chromatography, adsorption chromatography, and ultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described herein can be cultured to produce and/or secrete the biosynthetic products of the invention. For e, the alkene producers can be cultured for the biosynthetic tion of alkene.

For the production of alkene, the recombinant strains are cultured in a medium with carbon source and other essential nutrients. It is sometimes desirable and can be highly desirable to maintain anaerobic conditions in the fermenter to reduce the cost of the overall process. Such conditions can be obtained, for example, by first sparging the medium with en and then sealing the flasks with a septum and crimp-cap. For strains where growth is not observed anaerobically, microaerobic or substantially anaerobic conditions can be applied by perforating the septum with a small hole for W0 2013/040383 32 PCT/U82012/055469 limited aeration. Exemplary anaerobic conditions have been described previously and are well-known in the art. Exemplary aerobic and anaerobic conditions are described, for example, in United State publication 2009/0047719, filed August 10, 2007. Fermentations can be performed in a batch, fed-batch or uous manner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, in particular neutral pH, such as a pH of around 7 by addition of a base, such as NaOH or other bases, or acid, as needed to in the culture medium at a desirable pH. The growth rate can be determined by measuring optical density using a ophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time.

[0078] The growth medium can include, for example, any carbohydrate source which can supply a source of carbon to the non-naturally ing microorganism. Such sources include, for example, sugars such as glucose, xylose, arabinose, galactose, e, fructose, sucrose and starch. Other sources of ydrate include, for example, renewable feedstocks and biomass. Exemplary types of biomasses that can be used as feedstocks in the methods of the invention include cellulosic biomass, hemicellulosic biomass and lignin feedstocks or portions of feedstocks. Such s feedstocks contain, for example, carbohydrate substrates useful as carbon sources such as glucose, xylose, arabinose, ose, mannose, se and starch. Given the teachings and guidance provided herein, those skilled in the art will understand that renewable feedstocks and biomass other than those exemplified above also can be used for culturing the microbial organisms of the invention for the production of alkene.

In addition to renewable feedstocks such as those exemplified above, the alkene ial organisms of the invention also can be modified for growth on syngas as its source of carbon. In this c embodiment, one or more proteins or enzymes are sed in the alkene producing organisms to provide a metabolic y for utilization of syngas or other gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the major product of gasification of coal and of carbonaceous materials such as biomass materials, including agricultural crops and residues. Syngas is a mixture primarily of H2 and CO and can be ed from the gasification of any organic feedstock, including but not limited to coal, coal oil, natural gas, biomass, and waste organic matter. Gasification is generally carried out under a high filel to oxygen ratio. Although largely H2 and CO, syngas can also W0 2013/040383 33 include CO2 and other gases in smaller quantities. Thus, synthesis gas provides a cost effective source of gaseous carbon such as CO and, additionally, C02.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H2 to acetyl-CoA and other products such as acetate. Organisms capable of utilizing CO and syngas also generally have the capability of utilizing CO2 and CO2/H2 mixtures through the same basic set of enzymes and transformations assed by the Wood-Ljungdahl pathway. H2-dependent sion of CO2 to acetate by microorganisms was recognized long before it was revealed that CO also could be used by the same organisms and that the same pathways were involved. Many acetogens have been shown to grow in the presence of CO2 and produce compounds such as acetate as long as hydrogen is t to supply the necessary reducing equivalents (see for example, Drake, enesz’s, pp. 3-60 Chapman and Hall, New York, (1994)). This can be summarized by the following equation: 2 CO2 +4 H2+nADP+nPi—> CH3COOH+2H2O+nATP

[0082] Hence, non-naturally occurring microorganisms possessing the Wood- Ljungdahl pathway can utilize CO2 and H2 es as well for the production of acetyl- CoA and other d products.

The Wood-Ljungdahl y is well known in the art and consists of 12 reactions which can be separated into two branches: (1) methyl branch and (2) carbonyl branch. The methyl branch converts syngas to methyl-tetrahydrofolate (methyl-THF) s the carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in the methyl branch are catalyzed in order by the following enzymes or proteins: ferredoxin oxidoreductase, e dehydrogenase, formyltetrahydrofolate tase, methenyltetrahydrofolate cyclodehydratase, methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolate reductase. The reactions in the yl branch are catalyzed in order by the following s or proteins: methyltetrahydrofolate:corrinoid protein methyltransferase (for example, AcsE), corrinoid iron-sulfur protein, nickel-protein ly protein (for example, AcsF), ferredoxin, acetyl-CoA synthase, carbon monoxide dehydrogenase and nickel-protein assembly protein (for example, CooC). Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate an alkene y, those skilled in the art will understand that the same engineering design also can be performed with respect to introducing at least the W0 2013/040383 34 nucleic acids encoding the Wood-Ljungdahl enzymes or proteins absent in the host organism. ore, introduction of one or more ng nucleic acids into the microbial organisms of the invention such that the modified sm contains the complete Wood-Ljungdahl pathway will confer syngas utilization ability.

Additionally, the reductive (reverse) tricarboxylic acid cycle coupled with carbon monoxide dehydrogenase and/or hydrogenase ties can also be used for the conversion of C0, C02 and/or H2 to -CoA and other products such as acetate.

Organisms capable of fixing carbon via the reductive TCA pathway can utilize one or more of the following enzymes: ATP citrate-lyase, citrate lyase, aconitase, isocitrate dehydrogenase, ketoglutarate:ferredoxin oxidoreductase, succinyl-CoA synthetase, succinyl-CoA transferase, filmarate reductase, fumarase, malate dehydrogenase, NAD(P)H:ferredoxin oxidoreductase, carbon monoxide dehydrogenase, and hydrogenase.

Specifically, the reducing equivalents extracted from C0 and/or H2 by carbon monoxide dehydrogenase and hydrogenase are utilized to fix CO2 via the reductive TCA cycle into -CoA or acetate. Acetate can be converted to acetyl-CoA by enzymes such as acetyl-CoA erase, acetate kinase/phosphotransacetylase, and acetyl-CoA synthetase.

Acetyl-CoA can be converted to the alkene precursors, glyceraldehydephosphate, phosphoenolpyruvate, and pyruvate, by pyruvate:ferredoxin oxidoreductase and the enzymes of eogenesis. Following the teachings and guidance provided herein for introducing a sufficient number of encoding nucleic acids to generate an alkene pathway, those skilled in the art will understand that the same ering design also can be performed with respect to introducing at least the nucleic acids encoding the reductive TCA pathway enzymes or proteins absent in the host sm. Therefore, introduction of one or more encoding nucleic acids into the microbial organisms of the invention such that the modified organism contains a reductive TCA pathway can confer syngas utilization Accordingly, given the ngs and guidance provided herein, those skilled in the art will understand that a non-naturally occurring microbial organism can be produced that secretes the biosynthesized compounds of the invention when grown on a carbon source such as a ydrate. Such compounds include, for example, alkene and any of the intermediate metabolites in the alkene pathway. All that is required is to engineer in one or more of the required enzyme or protein activities to achieve biosynthesis of the desired compound or intermediate including, for e, inclusion of some or all of the alkene biosynthetic pathways. Accordingly, the invention es a non-naturally occurring microbial organism that produces and/or secretes alkene when grown on a carbohydrate or other carbon source and produces and/or secretes any of the intermediate metabolites shown in the alkene pathway when grown on a carbohydrate or other carbon source. The alkene producing microbial organisms of the ion can initiate synthesis from an intermediate, for example, an alkyl phosphate or an alkyl diphosphate.

The non-naturally occurring ial organisms of the invention are constructed using methods well known in the art as exemplified herein to exogenously express at least one nucleic acid encoding an alkene pathway enzyme or protein in sufficient amounts to produce alkene. It is understood that the microbial organisms of the invention are cultured under conditions sufficient to produce alkene. Following the teachings and guidance provided herein, the non-naturally occurring microbial organisms of the invention can achieve biosynthesis of alkene resulting in intracellular concentrations between about 01-200 mM or more. Generally, the intracellular concentration of alkene is between about 3-150 mM, particularly n about 5-125 mM and more particularly between about 8-100 mM, including about 10 mM, 20 mM, 50 mM, 80 mM, or more. ellular trations between and above each of these exemplary ranges also can be achieved from the turally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic or substantially bic growth or maintenance conditions. Exemplary anaerobic conditions have been described previously and are well known in the art. Exemplary anaerobic conditions for fermentation processes are described herein and are described, for example, in US. publication 047719, filed August 10, 2007. Any of these conditions can be employed with the non-naturally occurring microbial organisms as well as other anaerobic conditions well known in the art. Under such anaerobic or substantially anaerobic conditions, the alkene ers can synthesize alkene at intracellular trations of 5- mM or more as well as all other concentrations exemplified herein. It is understood that, even though the above description refers to intracellular concentrations, alkene producing microbial organisms can produce alkene intracellularly and/or secrete the product into the culture medium.

In addition to the ing and tation conditions disclosed herein, growth condition for achieving biosynthesis of alkene can include the addition of an osmoprotectant to the culturing ions. In certain embodiments, the non-naturally W0 2013/040383 36 PCT/U82012/055469 occurring microbial organisms of the invention can be sustained, cultured or fermented as described herein in the presence of an osmoprotectant. Briefly, an osmoprotectant refers to a compound that acts as an osmolyte and helps a microbial organism as described herein survive osmotic stress. otectants include, but are not limited to, betaines, amino acids, and the sugar ose. Non-limiting examples of such are glycine betaine, praline betaine, dimethylthetin, dimethylslfonioproprionate, 3-dimethylsulfonio methylproprionate, lic acid, ylsulfonioacetate, choline, tine and e. In one aspect, the osmoprotectant is glycine e. It is understood to one of ordinary skill in the art that the amount and type of osmoprotectant suitable for protecting a microbial organism described herein from osmotic stress will depend on the microbial organism used. The amount of osmoprotectant in the culturing conditions can be, for example, no more than about 0.1 mM, no more than about 0.5 mM, no more than about 1.0 mM, no more than about 1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no more than about 3.0 mM, no more than about 5.0 mM, no more than about 7.0 mM, no more than about lOmM, no more than about 50mM, no more than about 100mM or no more than about 500mM.

In some embodiments, the carbon feedstock and other cellular uptake s such as phosphate, ammonia, sulfate, chloride and other halogens can be chosen to alter the isotopic distribution of the atoms present in an alkene or any alkene pathway intermediate. The various carbon feedstock and other uptake sources enumerated above will be referred to herein, collectively, as “uptake s.” Uptake sources can provide isotopic enrichment for any atom present in the product alkene or alkene pathway intermediate, or for side products generated in reactions diverging away from an alkene pathway. Isotopic enrichment can be achieved for any target atom including, for example, carbon, hydrogen, oxygen, nitrogen, , phosphorus, chloride or other halogens.

In some embodiments, the uptake sources can be selected to alter the carbon- l2, carbon-l3, and carbon-l4 ratios. In some embodiments, the uptake sources can be selected to alter the oxygen-l6, oxygen-l7, and oxygen-18 ratios. In some embodiments, the uptake sources can be selected to alter the hydrogen, deuterium, and m . In some embodiments, the uptake sources can be selected to alter the nitrogen-l4 and nitrogen-15 ratios. In some embodiments, the uptake sources can be ed to alter the sulfur-32, sulfiJr-33, sulfur-34, and -35 ratios. In some embodiments, the uptake sources can be selected to alter the phosphorus-31, phosphorus-32, and phosphorus-33 ratios. In some embodiments, the uptake sources can be ed to alter the chlorine-35, ne-36, and chlorine-37 ratios.

In some embodiments, the isotopic ratio of a target atom can be varied to a desired ratio by selecting one or more uptake sources. An uptake source can be derived from a natural source, as found in nature, or from a man-made source, and one skilled in the art can select a natural source, a man-made source, or a combination thereof, to achieve a desired isotopic ratio of a target atom. An example of a man-made uptake source includes, for example, an uptake source that is at least partially d from a chemical synthetic reaction. Such isotopically ed uptake sources can be purchased commercially or prepared in the laboratory and/or optionally mixed with a natural source of the uptake source to e a desired isotopic ratio. In some embodiments, a target atom isotopic ratio of an uptake source can be achieved by selecting a d origin of the uptake source as found in nature. For example, as discussed herein, a natural source can be a biobased derived from or synthesized by a ical organism or a source such as petroleum-based products or the atmosphere. In some such embodiments, a source of carbon, for example, can be selected from a fossil fuel-derived carbon source, which can be relatively depleted of carbon-l4, or an environmental or atmospheric carbon source, such as C02, which can s a larger amount of carbon-l4 than its petroleum-derived counterpart.

[0092] The le carbon e carbon-l4 or radiocarbon makes up for roughly 1 in 1012 carbon atoms in the earth's atmosphere and has a half-life of about 5700 years. The stock of carbon is replenished in the upper atmosphere by a nuclear reaction involving cosmic rays and ordinary nitrogen (MN). Fossil fuels contain no carbon-l4, as it decayed long ago. Burning of fossil filels lowers the atmospheric carbon-l4 fraction, the so-called “Suess effect”.

Methods of determining the isotopic ratios of atoms in a compound are well known to those skilled in the art. Isotopic enrichment is readily assessed by mass spectrometry using techniques known in the art such as rated mass spectrometry (AMS), Stable Isotope Ratio Mass Spectrometry (SIRMS) and Site-Specific Natural Isotopic onation by Nuclear Magnetic Resonance (SNIF-NMR). Such mass spectral techniques can be ated with separation ques such as liquid chromatography (LC), high performance liquid chromatography (HPLC) and/or gas chromatography, and the like.

W0 2013/040383 38 2012/055469 In the case of carbon, ASTM D6866 was developed in the United States as a standardized analytical method for determining the biobased content of solid, liquid, and gaseous samples using radiocarbon dating by the American Society for Testing and Materials (ASTM) International. The standard is based on the use of radiocarbon dating for the determination of a product's biobased content. ASTM D6866 was first published in 2004, and the current active version of the standard is ASTM D6866-11 (effective April 1, 2011). Radiocarbon dating techniques are well known to those skilled in the art, including those described herein.

The biobased content of a compound is estimated by the ratio of carbon-14 (14C) to carbon-12 (12C). Specifically, the Fraction Modern (Fm) is computed from the expression: Fm = (S-B)/(M-B), where B, S and M represent the C ratios of the blank, the sample and the modern reference, respectively. on Modern is a measurement of the deviation of the 14C/12C ratio of a sample from "Modern." Modern is defined as 95% of the radiocarbon concentration (in AD 1950) of National Bureau of Standards (NBS) Oxalic Acid 1 (i.e., standard reference materials (SRM) 4990b) normalized to 513CVPDB=- 19 per mil (Olsson, The use 0f0xalz'c acid as a Standard. in, arbon Variations and Absolute Chronology, Nobel Symposium, 12th Proc., John Wiley & Sons, New York ). Mass spectrometry results, for example, measured by ASM, are calculated using the internationally agreed upon definition of 0.95 times the specific activity ofNBS Oxalic Acid I (SRM 4990b) ized to 813CVPDB=-l9 per mil. This is equivalent to an absolute (AD 1950) C ratio of 1.176 0.010 x 10‘12 (Karlen et al., Arkz'v Geofysz'k, 4:465-471 (1968)). The standard calculations take into account the ential uptake of one istope with respect to another, for example, the preferential uptake in biological l2 l3 l4 - systems of C over C over C and these corrections are ed as a Fm corrected for 8 .

An oxalic acid standard (SRM 4990b or HOx 1) was made from a crop of 1955 sugar beet. Although there were 1000 lbs made, this oxalic acid standard is no longer commercially ble. The Oxalic Acid 11 standard (HOx 2; T designation SRM 4990 C) was made from a crop of 1977 French beet molasses. In the early 1980's, a group of 12 laboratories measured the ratios of the two standards. The ratio of the activity of Oxalic acid 11 to 1 is 1.2933::0.001 (the ed mean). The isotopic ratio of HOx II is - 17.8 per mille. ASTM D6866-11 suggests use of the available Oxalic Acid 11 standard SRM 4990 C (Hox2) for the modern standard (see discussion of original vs. currently W0 2013/040383 39 available oxalic acid standards in Mann, arbon, 25(2):519-527 (1983)). A Fm = 0% represents the entire lack of carbon-14 atoms in a material, thus ting a fossil (for example, petroleum based) carbon source. A Fm = 100%, after correction for the post- 1950 injection of carbon-14 into the atmosphere from nuclear bomb testing, indicates an entirely modern carbon source. As described herein, such a “modern” source includes biobased sources.

As described in ASTM D6866, the percent modern carbon (pMC) can be greater than 100% because of the continuing but shing effects of the 1950s nuclear testing programs, which resulted in a considerable enrichment of -14 in the atmosphere as described in ASTM D6866-11. Because all sample carbon-14 activities are referenced to a “pre-bomb” rd, and because nearly all new biobased products are produced in a post-bomb environment, all pMC values (after correction for isotopic fraction) must be multiplied by 0.95 (as of 2010) to better reflect the true ed t of the sample. A biobased t that is greater than 103% ts that either an analytical error has occurred, or that the source of biobased carbon is more than several years old.

ASTM D6866 quantifies the biobased content relative to the material’s total organic content and does not consider the inorganic carbon and other non-carbon containing substances present. For example, a product that is 50% starch-based material and 50% water would be considered to have a Biobased t = 100% (50% organic content that is 100% biobased) based on ASTM D6866. In another example, a product that is 50% starch-based material, 25% petroleum-based, and 25% water would have a ed Content = 66.7% (75% organic content but only 50% of the product is biobased). In another example, a product that is 50% organic carbon and is a petroleum- based product would be considered to have a Biobased t = 0% (50% organic carbon but from fossil sources). Thus, based on the well known methods and known standards for determining the biobased content of a compound or material, one d in the art can y determine the biobased content and/or prepared downstream products that utilize of the invention having a desired biobased content.

[0099] Applications of carbon-14 dating ques to quantify bio-based content of materials are known in the art e et al., Nuclear Instruments and Methods in Physics Research B, 172:281-287 (2000)). For example, carbon-14 dating has been used to quantify bio-based content in terephthalate-containing materials (Colonna et al., Green W0 2013/040383 40 2012/055469 Chemistry, 13:2543-2548 (2011)). Notably, polypropylene terephthalate (PPT) polymers derived from renewable opanediol and petroleum-derived terephthalic acid resulted in Fm values near 30% (i.e., since 3/11 of the polymeric carbon derives from renewable 1,3-propanediol and 8/11 from the fossil end member terephthalic acid) e et al., supra, 2000). In contrast, polybutylene thalate polymer derived from both renewable 1,4-butanediol and renewable terephthalic acid resulted in bio-based content ing 90% (Colonna et al., supra, 2011).

Accordingly, in some ments, the present invention provides an alkene or an alkene pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects an atmospheric carbon, also referred to as environmental carbon, uptake source. For example, in some aspects the alkene or alkene pathway intermediate can have an Fm value of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least %, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or as much as 100%. In some such embodiments, the uptake source is C02. In some embodiments, the present invention provides an alkene or an alkene pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that reflects petroleum-based carbon uptake source. In this aspect, the alkene or alkene pathway intermediate can have an Fm value of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% or less than 1%. In some embodiments, the present ion provides an alkene or an alkene pathway intermediate that has a carbon-12, carbon-13, and carbon-14 ratio that is obtained by a combination of an atmospheric carbon uptake source with a petroleum-based uptake source. Using such a combination of uptake s is one way by which the carbon-12, carbon-13, and carbon-14 ratio can be varied, and the respective ratios would reflect the proportions of the uptake sources.

Further, the present invention relates to the biologically produced alkene or alkene pathway intermediate as disclosed herein, and to the products derived therefrom, wherein the alkene or alkene y ediate has a carbon-12, carbon-13, and carbon-14 isotope ratio of about the same value as the C02 that occurs in the environment.

For example, in some aspects the invention es bioderived alkene or a bioderived alkene intermediate having a carbon-12 versus carbon-13 versus carbon-14 isotope ratio of W0 2013/040383 41 PCT/U82012/055469 about the same value as the C02 that occurs in the environment, or any of the other ratios disclosed herein. It is understood, as sed herein, that a product can have a carbon-12 versus carbon-l3 versus carbon-l4 isotope ratio of about the same value as the C02 that occurs in the environment, or any of the ratios disclosed herein, wherein the product is generated from a bioderived alkene or a bioderived alkene pathway intermediate as disclosed herein, wherein the ived product is chemically modified to generate a final t. Methods of chemically modifying a bioderived product of alkene, or an intermediate thereof, to generate a desired product are well known to those skilled in the art, as described herein. The invention further provides a plastic, a polymer, a co-polymer, a polymer intermediate, a resin, a rubber, or a fiber having a carbon-12 versus carbon-l3 versus -l4 isotope ratio of about the same value as the C02 that occurs in the environment, wherein the a plastic, a polymer, a co-polymer, a polymer intermediate, a resin, a rubber, or a fiber are generated ly from or in combination with bioderived alkene or a bioderived alkene pathway intermediate as disclosed herein.

[00102] Alkenes include a variety chemicals as described herein, which can be used in commercial and industrial applications. For example, the alkenes disclosed herein can be used as a raw material in the production of a wide range of products including plastics, polymers, ymers, polymer intermediates, resins, rubbers, or fibers. Accordingly, in some embodiments, the invention provides biobased plastics, polymers, ymers, polymer intermediates, resins, rubbers, or fibers sing one or more bioderived alkene or bioderived alkene pathway intermediate produced by a non-naturally occurring microorganism of the invention or produced using a method disclosed herein.

As used herein, the term "bioderived" means d from or synthesized by a biological organism and can be considered a ble resource since it can be ted by a ical organism. Such a biological organism, in particular the microbial organisms of the invention disclosed herein, can e feedstock or s, such as, sugars or carbohydrates obtained from an agricultural, plant, bacterial, or animal source.

Alternatively, the biological organism can utilize atmospheric carbon. As used herein, the term sed" means a product as described above that is composed, in whole or in part, of a bioderived compound of the ion. A biobased or ived product is in contrast to a petroleum derived product, n such a product is derived from or synthesized from petroleum or a petrochemical feedstock.

W0 2013/040383 42 PCT/U82012/055469 In some ments, the invention provides a plastic, a polymer, a co- polymer, a polymer intermediate, a resin, a rubber, or a fiber comprising a bioderived alkene or a bioderived alkene pathway intermediate, wherein the bioderived alkene or bioderived alkene pathway intermediate includes all or part of the alkene or alkene pathway intermediate used in the production of the plastic, polymer, co-polymer, polymer intermediate, resin, , or fiber. Thus, in some aspects, the invention provides a biobased plastic, polymer, co-polymer, polymer intermediate, resin, rubber, or fiber comprising at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% bioderived alkene or bioderived alkene pathway ediate as disclosed . Additionally, in some aspects, the ion provides a ed plastic, polymer, co-polymer, polymer intermediate, resin, , or fiber wherein the alkene or alkene y intermediate used in its production is a combination of bioderived and eum derived alkene or alkene pathway intermediate. For example, a biobased plastic, polymer, co-polymer, polymer intermediate, resin, rubber, or fiber can be produced using 50% bioderived alkene and 50% eum derived alkene or other desired ratios such as 60%/40%, 70%/30%, 80%/20%, 90%/10%, 95%/5%, 100%/0%, 40%/60%, 30%/70%, 20%/80%, 10%/90% of bioderived/petroleum derived precursors, so long as at least a portion of the product comprises a bioderived product produced by the microbial organisms disclosed . It is understood that methods for producing plastic, polymer, co-polymer, polymer intermediate, resin, rubber, or fiber using the bioderived alkene or bioderived alkene pathway intermediate of the ion are well known in the art.

The culture conditions can include, for example, liquid culture procedures as well as fermentation and other large scale culture procedures. As described herein, particularly useful yields of the biosynthetic products of the invention can be obtained under anaerobic or substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achieving thesis of alkene includes anaerobic culture or fermentation conditions. In certain embodiments, the non-naturally ing microbial organisms of the invention can be sustained, cultured or fermented under anaerobic or ntially anaerobic ions.

Briefly, anaerobic ions refers to an environment devoid of oxygen. Substantially anaerobic conditions include, for example, a culture, batch fermentation or continuous W0 2013/040383 43 PCT/U82012/055469 fermentation such that the dissolved oxygen concentration in the medium remains between 0 and 10% of saturation. ntially anaerobic conditions also includes growing or resting cells in liquid medium or on solid agar inside a sealed chamber maintained with an atmosphere of less than 1% oxygen. The percent of oxygen can be maintained by, for example, sparging the culture with an Nz/COZ mixture or other suitable non-oxygen gas or gases.

The culture conditions bed herein can be scaled up and grown continuously for manufacturing of alkene. Exemplary growth procedures include, for example, tch fermentation and batch separation; fed-batch fermentation and continuous separation, or uous fermentation and continuous separation. All of these processes are well known in the art. Fermentation procedures are particularly useful for the biosynthetic production of commercial quantities of . Generally, and as with non-continuous culture procedures, the continuous and/or near-continuous production of alkene will include culturing a non-naturally occurring alkene ing organism of the invention in ient nutrients and medium to sustain and/or nearly sustain growth in an exponential phase. Continuous culture under such conditions can e, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally, continuous culture can include longer time periods of 1 week, 2, 3, 4 or 5 or more weeks and up to several months. Alternatively, organisms of the invention can be cultured for hours, if suitable for a particular ation. It is to be understood that the continuous and/or near-continuous culture conditions also can include all time intervals in between these ary periods.

It is further understood that the time of culturing the microbial sm of the invention is for a sufficient period of time to produce a sufficient amount of product for a desired purpose.

[00108] tation procedures are well known in the art. Briefly, fermentation for the biosynthetic production of alkene can be ed in, for example, fed-batch fermentation and batch tion; fed-batch fermentation and continuous separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

[00109] In addition to the above fermentation procedures using the alkene producers of the invention for continuous tion of substantial quantities of alkene, the alkene producers also can be, for e, simultaneously subjected to chemical synthesis procedures to convert the product to other compounds or the product can be separated W0 2013/040383 44 PCT/U82012/055469 from the fermentation e and sequentially subjected to chemical or enzymatic conversion to convert the product to other compounds, if desired.

To generate better producers, metabolic modeling can be utilized to optimize growth conditions. Modeling can also be used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, US. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and US. Patent No. 7,127,379). ng analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of alkene.

[00111] One computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired t is the OptKnock computational framework (Burgard et al., Biotechnol. Bioeng. 84:647-657 (2003)). OptKnock is a metabolic ng and simulation m that suggests gene deletion or disruption strategies that result in genetically stable rganisms which overproduce the target product.

Specifically, the framework examines the te metabolic and/or biochemical network of a microorganism in order to suggest genetic manipulations that force the desired biochemical to become an obligatory byproduct of cell growth. By ng biochemical production with cell growth through strategically placed gene ons or other functional gene disruption, the growth selection res imposed on the engineered strains after long periods of time in a bioreactor lead to improvements in performance as a result of the compulsory growth-coupled biochemical production. Lastly, when gene deletions are constructed there is a negligible possibility of the designed strains ing to their wild- type states e the genes selected by OptKnock are to be completely removed from the genome. Therefore, this computational methodology can be used to either fy alternative pathways that lead to biosynthesis of a desired product or used in connection with the non-naturally occurring microbial organisms for further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computational method and system for modeling cellular metabolism. The OptKnock m relates to a framework of models and methods that incorporate particular constraints into flux balance analysis (FBA) . These aints include, for example, qualitative kinetic information, qualitative regulatory information, and/or DNA rray experimental data. OptKnock also computes solutions to s metabolic problems by, for example, tightening the flux 45 2012/055469 boundaries derived through flux balance models and subsequently probing the performance limits of metabolic networks in the presence of gene additions or ons.

OptKnock computational ork allows the construction of model formulations that allow an effective query of the performance limits of metabolic networks and provides methods for solving the resulting mixed-integer linear programming problems. The metabolic modeling and simulation methods referred to herein as OptKnock are described in, for example, US. publication 168654, filed January 10, 2002, in International Patent No. 02/00660, filed January 10, 2002, and US. publication 2009/0047719, filed August 10, 2007.

[00113] Another computational method for identifying and designing metabolic alterations favoring biosynthetic production of a product is a metabolic modeling and simulation system termed SimPheny®. This computational method and system is described in, for example, US. publication 2003/0233218, filed June 14, 2002, and in International Patent Application No. PCT/US03/l 8838, filed June 13, 2003. SimPheny® is a ational system that can be used to e a network model in silica and to simulate the flux of mass, energy or charge through the chemical reactions of a biological system to define a solution space that contains any and all possible functionalities of the chemical reactions in the system, thereby ining a range of allowed activities for the ical system. This approach is referred to as constraints-based modeling because the solution space is defined by constraints such as the known iometry of the included reactions as well as reaction dynamic and capacity aints associated with maximum fluxes through reactions. The space defined by these constraints can be interrogated to determine the phenotypic capabilities and behavior of the ical system or of its biochemical components.

[00114] These computational ches are consistent with biological realities because biological systems are flexible and can reach the same result in many different ways. Biological systems are designed through evolutionary mechanisms that have been restricted by fundamental constraints that all living systems must face. Therefore, constraints-based modeling strategy embraces these general realities. Further, the ability to continuously impose further restrictions on a network model via the tightening of constraints results in a reduction in the size of the solution space, y enhancing the precision with which physiological performance or phenotype can be predicted.

W0 40383 46 PCT/U82012/055469 Given the teachings and ce provided herein, those d in the art will be able to apply various ational frameworks for metabolic modeling and simulation to design and implement biosynthesis of a desired nd in host microbial organisms.

Such metabolic modeling and simulation methods e, for example, the computational systems exemplified above as SimPheny® and OptKnock. For illustration of the ion, some methods are described herein with reference to the OptKnock ation framework for modeling and simulation. Those skilled in the art will know how to apply the identification, design and implementation of the metabolic alterations using OptKnock to any of such other metabolic modeling and simulation ational frameworks and methods well known in the art.

The methods described above will provide one set of metabolic reactions to disrupt. Elimination of each on within the set or metabolic modification can result in a desired product as an obligatory product during the growth phase of the organism.

Because the reactions are known, a solution to the bilevel OptKnock problem also will provide the associated gene or genes ng one or more enzymes that catalyze each reaction within the set of ons. Identification of a set of reactions and their corresponding genes encoding the enzymes participating in each reaction is generally an automated process, accomplished through correlation of the reactions with a reaction se having a onship between enzymes and encoding genes.

[00117] Once identified, the set of reactions that are to be disrupted in order to achieve production of a desired product are implemented in the target cell or organism by functional disruption of at least one gene encoding each metabolic reaction within the set.

One particularly useful means to achieve functional disruption of the reaction set is by deletion of each encoding gene. However, in some instances, it can be beneficial to disrupt the reaction by other genetic aberrations including, for example, mutation, on of regulatory regions such as promoters or cis binding sites for regulatory factors, or by truncation of the coding sequence at any of a number of locations. These latter aberrations, resulting in less than total deletion of the gene set can be useful, for example, when rapid assessments of the coupling of a product are desired or when genetic reversion is less likely to occur.

To identify additional productive solutions to the above described bilevel OptKnock problem which lead to further sets of reactions to disrupt or metabolic ations that can result in the biosynthesis, including growth-coupled biosynthesis of a desired product, an zation method, termed integer cuts, can be implemented. This method proceeds by iteratively solving the OptKnock problem exemplified above with the incorporation of an additional constraint referred to as an integer cut at each iteration.

Integer cut constraints effectively prevent the solution procedure from choosing the exact same set of reactions identified in any previous ion that obligatorily couples product biosynthesis to growth. For e, if a previously identified -coupled metabolic modification specifies reactions 1, 2, and 3 for disruption, then the following constraint prevents the same reactions from being simultaneously ered in subsequent solutions.

The integer cut method is well known in the art and can be found described in, for example, Burgard et al., Biotechnol. Prog. 17:791-797 (2001). As with all methods described herein with reference to their use in ation with the OptKnock computational framework for metabolic modeling and simulation, the integer cut method of reducing redundancy in iterative computational analysis also can be applied with other computational frameworks well known in the art including, for example, SimPheny®.

[00119] The s exemplified herein allow the construction of cells and organisms that thetically produce a desired product, including the obligatory coupling of production of a target mical product to growth of the cell or organism engineered to harbor the identified genetic alterations. Therefore, the ational methods described herein allow the identification and implementation of metabolic modifications that are fied by an in silico method selected from ck or SimPheny®. The set of metabolic modifications can include, for example, addition of one or more biosynthetic pathway enzymes and/or functional disruption of one or more metabolic reactions including, for example, disruption by gene deletion.

As discussed above, the OptKnock ology was developed on the premise that mutant microbial networks can be evolved towards their computationally predicted maximum-growth phenotypes when subjected to long periods of growth selection. In other words, the approach ges an organism's ability to self-optimize under selective pressures. The OptKnock framework allows for the exhaustive enumeration of gene deletion combinations that force a coupling between biochemical production and cell growth based on network stoichiometry. The identification of optimal gene/reaction knockouts requires the on of a bilevel optimization m that chooses the set of active reactions such that an optimal growth solution for the resulting network W0 2013/040383 48 overproduces the biochemical of interest (Burgard et al., Biotechnol. Bioeng. -657 (2003)).

An in silico stoichiometric model of E. coli lism can be employed to identify essential genes for metabolic pathways as exemplified previously and bed in, for example, US. patent publications US 012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in US. Patent No. 7,127,379. As disclosed herein, the OptKnock mathematical framework can be applied to pinpoint gene deletions leading to the growth- coupled production of a desired product. r, the solution of the bilevel OptKnock problem provides only one set of deletions. To enumerate all meaningful solutions, that is, all sets of knockouts leading to growth-coupled tion formation, an optimization technique, termed integer cuts, can be ented. This entails iteratively solving the OptKnock problem with the incorporation of an onal constraint referred to as an integer cut at each iteration, as discussed above.

[00122] As disclosed herein, a nucleic acid encoding a desired activity of an alkene pathway can be introduced into a host organism. In some cases, it can be desirable to modify an activity of an alkene pathway enzyme or protein to se production of alkene. For example, known mutations that increase the activity of a protein or enzyme can be uced into an encoding nucleic acid molecule. Additionally, optimization methods can be applied to se the activity of an enzyme or protein and/or decrease an tory activity, for example, decrease the activity of a negative regulator.

One such optimization method is directed evolution. Directed evolution is a powerful approach that involves the introduction of ons ed to a specific gene in order to improve and/or alter the properties of an enzyme. Improved and/or altered enzymes can be identified through the development and implementation of sensitive high- throughput ing assays that allow the automated screening ofmany enzyme variants (for example, >104). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme with zed properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme variants that need to be generated and screened. Numerous directed evolution technologies have been developed (for reviews, see Hibbert et al., BiomalEng 22: 1 1-19 (2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC W0 2013/040383 49 PCT/U82012/055469 Press; Otten and Quax. .Eng 22:1-9 (2005).; and Sen et al., Appl Biochem.Bi0tec/m0[ 143:212-223 (2007)) to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme classes. Enzyme characteristics that have been improved and/or altered by directed evolution technologies include, for example: selectivity/specificity, for conversion of non-natural ates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH ions; substrate or product nce, so that high product titers can be achieved; binding (Km), including broadening ate g to include non-natural substrates; tion (K), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increases enzymatic on rates to achieve desired flux; expression levels, to increase protein yields and overall pathway flux; oxygen stability, for operation of air sensitive enzymes under c conditions; and anaerobic activity, for operation of an aerobic enzyme in the absence of oxygen.

[00124] A number of exemplary methods have been developed for the nesis and diversification of genes to target desired properties of specific s. Such methods are well known to those skilled in the art. Any of these can be used to alter and/or optimize the activity of an alkene pathway enzyme or n. Such methods include, but are not limited to EpPCR, which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (Pritchard et al., J Thear.Bi0[. 234:497-509 (2005)); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole ar plasmid is used as the template and random 6-mers with lease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res. 32:el45 (2004); and Fujii et al., Nat. Pr0t0c. 12493-2497 (2006)); DNA or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc Natl Acad Sci USA 91 :10747-10751 (1994); and r, Nature 370:389-391 (1994)); Staggered Extension (StEP), which entails template priming followed by repeated cycles of 2 step PCR with denaturation and very short duration of annealing/extension (as short as 5 sec) (Zhao et al., Nat. Biotechnol. 16:258-261 (1998)); Random Priming ination (RPR), in which random sequence primers are used to generate many short DNA fragments mentary to different segments of the template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).

Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form duplexes that are repaired by mismatch repair v et al, Nucleic Acids Res. 27:e18 ; and Volkov et al., Methods l. 328:456-463 (2000)); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol. 19:354-359 (2001)); Recombined ion on Truncated templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (Lee et al., J. Molec. sis 26:119-129 ); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist and Gibbs, Methods M0l.Bi0l 352: 191-204 (2007); Bergquist et al., Bi0m0l.Eng 22:63-72 (2005); Gibbs et al., Gene 271 :13-20 ); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair ons of a gene or gene fragment of interest (Ostermeier et al., Proc. Natl. Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat. Biotechnol. 17: 1205-1209 (1999)); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (Lutz et al., c Acids Res 29:E16 (2001)); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA 98: 1 1248-1 1253 (2001)); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening/selection for those retaining usable activity (Bergquist et al., Bi0m0l. Eng. 22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothioate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (Wong et al., hnol. J. 3:74-82 (2008); Wong et al., Nucleic Acids Res. 32:e26 ; and Wong et al., Anal. Biochem. 341 :187-189 (2005)); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode “all genetic ity in targets” and allows a very high diversity for the shuffled y (Ness et al., Nat. Biotechnol. : 125 1-1255 (2002)); Nucleotide ge and Excision Technology NexT, which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then dine to perform endpoint DNA fragmentation (Muller et al., Nucleic Acids Res. 33 :e1 17 (2005)).

Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, ing in libraries of single-crossover hybrids (Sieber et al., Nat. Biotechnol. 19:456-460 (2001)); Gene Site Saturation MutagenesisTM (GSSMTM), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations (Kretz et al., Methods Enzymol. 3883-11 (2004)); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of le amino acid sequence alterations (Reidhaar—Olson et al. Methods Enzymol. 208:564-586 (1991); and Reidhaar—Olson et al. e 241 :53-57 ); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (Reetz et al., Angew. Chem. Int. Ed Engl. 9-3591 ); the Mutator Strains technique, in which conditional ts mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit ofDNA polymerase III, to allow increases of 20 to 4000-X in random and natural mutation frequency during selection and block accumulation of deleterious mutations when ion is not required (Selifonova et al., Appl. Environ. Microbiol. 67:3645-3649 (2001)); Low et al., J. Mol. Biol. 260359-3680 ).

] Additional exemplary methods include Look-Through nesis (LTM), which is a imensional mutagenesis method that assesses and optimizes combinatorial ons of selected amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA 102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (Tunable GeneReassemblyTM (TGRTM) Technology supplied by Verenium ation), in Silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein ne possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein tics, and generally works most effectively on proteins WO 40383 52 with known three-dimensional ures (Hayes et al., Proc. Natl. Acad. Sci. USA 99: 15926-15931 (2002)); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure/function to choose a likely site for enzyme improvement, performing tion mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screening/selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired ty is ed (Reetz et al., Nat. Protoc. 2:891-903 ; and Reetz et al., Angew. Chem. Int. Ed Engl. 45:7745-7751 (2006)).

Any of the aforementioned methods for mutagenesis can be used alone or in any combination. Additionally, any one or combination of the directed evolution methods can be used in conjunction with ve evolution techniques, as described herein.

It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also provided within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Enzyme candidates for zing steps A-E of Figure 1 Alcohol kinase [Figure 1: Step A] Alcohol kinase enzymes ze the transfer of a phosphate group to a hydroxyl group. Kinases that catalyze transfer of a phosphate group to an alcohol group are members of the EC 2.7.1 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.1 enzyme class.

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H H H H H SN SN SN SN SN 56 2012/055469 Mevalonate kinase (EC 2.7.1.36) phosphorylates the al hydroxyl group of mevalonate. Gene candidates for this step include erg12 from S. cerevisiae, mvk from Methanocaldococcusjannaschi, MVK from Homo sapeins, and mvk from Arabidopsis thaliana col. Additional mevalonate kinase candidates e the feedback-resistant mevalonate kinase from the archeon Methanosarcina mazei (Primak et al, AEM, in press (2011)) and the ka protein from Streptococcus pneumoniae (Andreassi et al, Protein Sci, 16:983-9 (2007)). ka proteins from S. cerevisiae, S. pneumoniae and M. mazei were heterologously expressed and characterized in E. coli (Primak et al, supra). The S. pneumoniae mevalonate kinase was active on several alternate ates ing cylopropylmevalonate, vinylmevalonate and ethynylmevalonate (Kudoh et al, Bioorg Med Chem 18: 1 124-34 ), and a uent study determined that the ligand binding site is selective for compact, electron-rich C(3)—substituents (Lefurgy et al, JBiol Chem 285:20654-63 (2010)).

NP 8510841 30690651 Arabidosis thaliana NP 6337861 21227864 NP 3579321 15902382

[00132] Glycerol kinase also phosphorylates the terminal hydroxyl group in glycerol to form glycerolphosphate. This reaction occurs in several species, including Escherichia coli, romyces siae, and Therm0t0ga maritima. The E. coli glycerol kinase has been shown to accept alternate substrates such as dihydroxyacetone and glyceraldehyde (Hayashi et al., J Biol. Chem. 242:1030-1035 (1967)). T. maritime has two glycerol kinases (Nelson et al., Nature 399:323-329 (1999)). Glycerol kinases have been shown to have a wide range of ate specificity. Crans and Whiteside d glycerol kinases from four different organisms richia coli, S. cerevisiae, Bacillus stear0therm0philus, and Candida mycoderma) (Crans et al., J.Am.Chem.S0c. 107:7008-7018 (2010); Nelson et al., supra, (1999)). They studied 66 different analogs of glycerol and concluded that the enzyme could accept a range of substituents in place of one al hydroxyl group and that the hydrogen atom at C2 could be replaced by a methyl group. Interestingly, the kinetic constants of the enzyme from all four organisms were very similar.

GenBank ID GI Number W0 2013/040383 57 Protein GenBank ID GI Number glpK AP_003883.1 89110103 Escherichia coli K12 ; [1K1 NP 2287601 15642775 Therm0t0 a maritime MSB8 ; [1K2 NP 2292301 15642775 Therm0t0 a maritime MSB8 Gut] NP_011831.1 82795252 Homoserine kinase is r le candidate. This enzyme is also present in a number of organisms ing E. coli, Streptomyces sp, and S. cerevisiae.

Homoserine kinase from E. coli has been shown to have activity on numerous substrates, including, Lamino,l,4- butanediol, ate semialdehyde, and 2-amino hydroxyvalerate (Huo et al., Biochemistry 35: 16180-16185 (1996); Huo et al., i0chem.Bi0phys. 330:373-379 (1996)). This enzyme can act on substrates where the carboxyl group at the alpha position has been replaced by an ester or by a hydroxymethyl group. The gene candidates are: Protein GenBank ID GI Number thrB BAB96580.2 85674277 Escherichia coli K12 SACTlDRAFT_4809 ZP_06280784.1 282871792 Streptomyces sp. ACT-I ThrI 54.1 172978 Saccharomyces serevisiae Phosphate 1yase [Figure 1: Step B] ] Phosphate lyase enzymes catalyze the sion of alkyl phosphates to alkenes. Carbon-oxygen lyases that operate on phosphates are found in the EC 4.2.3 enzyme class. The table below lists several relevant enzymes in EC class 4.2.3.

Enz me Commission Number Enz me Name Chorismate s nthase 4.2.3.15 M rcene s nthase 4.2.3.26 Linalools nthase 4.2.3.27 Is0orene s nthase 4.2.3.36 Te oentriene s hase 4.2.3.46 E, E -aloha-Famesene s nthase 4.2.3.47 Beta-Farnesene s nthase 4.2.3.49 dol s nthase

[00135] Chorismate synthase (EC 4.2.3.5) participates in the shikimate pathway, zing the dephosphorylation of 5-enolpyruvylshikimatephosphate to chorismate.

The enzyme requires reduced flavin mononucleotide (FMN) as a cofactor, although the net reaction of the enzyme does not involve a redox change. In contrast to the enzyme found in plants and ia, the chorismate synthase in fungi is also able to reduce FMN at the expense ofNADPH roux et a1., Planta 207:325-334 ). Representative monofunctional enzymes are encoded by aroC ofE. coli (White et a1., Biochem. J. 251 :313-322 (1988)) and Streptococcus pneumoniae (Maclean and Ali, Structure 11:1499- 1511 (2003)). Bifunctional fiangal enzymes are found in Neurospora crassa (Kitzing et al., J. Biol. Chem. 276:42658-42666 (2001)) and Saccharomyces cerevisiae (Jones et a1., Mol. Microbiol. 52143-2152 (1991)).

GenBank , NP 4168321 16130264 Escherichia coli ACH47980.1 197205483 U25818.1.-19..1317 AAC49056.1 976375 AR02 CAA42745.1 3387 Isoprene synthase naturally catalyzes the conversion of dimethylallyl diphosphate to isoprene, but can also catalyze the synthesis of 1,3-butadiene from 2- butenyldiphosphate. Isoprene synthases can be found in several organisms including s alba (Sasaki et a1., FEBS Letters, 2005, 579 (11), 2514-2518), Pueraria montana (Lindberg et a1., Metabolic Eng, 12(1):70-79 (2010); Sharkey et al., Plant Physiol, 137(2):700-712 (2005)), and Populus tremula X Populus alba, also called Populus canescens r et al., Planta, 2001, 213 (3), 7). The crystal structure of the s canescens isoprene synthase was determined (Koksal et al, J Mol Biol 402363- 373 (2010)). Additional isoprene synthase enzymes are bed in (Chotani et a1., WO/2010/031079, Systems Using Cell Culture for Production of Isoprene; Cervin et a1., US Patent Application 20100003716, Isoprene Synthase Variants for Improved Microbial tion of Isoprene).

Protein k ID GI Number isS BAD98243.1 10 P0Iulus alba isS AAQ84170-1 35187004 isS 96.1 13539551 P0Iulus tremula x P0Iulus alba Myrcene synthase enzymes catalyze the dephosphorylation of geranyl diphosphate to yrcene (EC 4.2.3.15). Exemplary myrcene synthases are encoded by MST2 ofSolanum rsicum (van Schie et al, Plant Mol Biol 64:D473-79 (2007)), TPS-Myr of Picea abies (Martin et al, Plant Physiol 135:1908-27 (2004)) g-myr ofAbies grandis (Bohlmann et al, J Biol Chem 272:21784-92 (1997)) and TPSlO ofArabidopsis thaliana ann et al, Arch Biochem Biophys 375:261-9 (2000)). These enzymes were heterologously expressed in E. 0012'.

Protein GenBank ID GI Number MST2 ACN58229.1 224579303 TPS-M r AAS47690.2 77546864 G—m r 0244741 17367921 TPSIO 3.1 330252449 Arabidmsz's thaliana ] Famesyl diphosphate is converted to alpha-famesene and beta-famesene by alpha-famesene synthase and beta-famesene synthase, respectively. Exemplary alpha- famesene synthase enzymes e TPS03 and TPS02 of opsis thaliana (Faldt et al, Planta 216:745-51 (2003); Huang et al, Plant l 153: 1293-3 10 (2010)), afs of Cacamz's sativas (Mercke et al, Plant Physiol 135:2012-14 (2004), eafar ofMalas x domestica (Green et a1, Phytochem 68: 176-88 (2007)) and r of Picea abies (Martin, supra). An exemplary beta-famesene synthase enzyme is encoded by TPSI of Zea mays (Schnee et al, Plant Physiol 130:2049-60 (2002)).

Protein 713503 TPSO2 a s AAU05951.1 51537953 ea ar Q84L1322 75241161 TPSI Q84ZW8-1 75149279 Diphosphokinase (Figure 1: Step C1

[00139] Diphosphokinase enzymes catalyze the transfer of a diphosphate group to an alcohol group. The enzymes described below naturally possess such activity. Kinases that catalyze transfer of a phate group are members of the EC 2.7.6 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.6 enzyme class. 2.7 6 1 ribose-oohoshate dioohoshokinase 2.7 6 2 thiamine dioohoshokinase 2-aminohydroxyhydroxymethyldihydropteridine 2 7 6 3 dioohoshokinase 2.7 6 4 nucleotide diphosphokinase W0 2013/040383 60 _Enz me Commission No. Enz me Name 2.7.6.5 GTP di ohos ohokinase Of particular interest are ribose-phosphate diphosphokinase enzymes, which have been identified in Escherichia coli (Hove-Jenson et al., JBiol Chem, 1986, 26l(l5);6765-7l) and Mycoplasma pneumoniae M129 (McElwain et al, International Journal ofSystematic Bacteriology, 1988, 38:417-423) as well as thiamine diphosphokinase enzymes. Exemplary thiamine diphosphokinase enzymes are found in Arabidopsis thaliana i, Plant Mol Biol, 2007, 65(1-2);l5l-62).

GenBank ID GI Number _NP 4157251 16129170 Escherichia coli NP_109761. 1 13507812 Mycoplasmapneamoniae M129 TPK1 BAH19964.1 222424006 msis thaliana col BAH57065.1 22 7204427 Arabido sis thaliana col Alkyl phosphate kinase gFigure 1: Step D1

[00141] Alkyl phosphate kinase enzymes ze the transfer of a phosphate group to the phosphate group of an alkyl phosphate. The enzymes described below naturally possess such activity or can be engineered to exhibit this activity. Kinases that catalyze transfer of a phosphate group to another phosphate group are s of the EC 2.7.4 enzyme class. The table below lists several useful kinase enzymes in the EC 2.7.4 enzyme class.

Enz me Commission No. En me Name 2.7.4.1 ool ohos hate kinase 2.7.4.2 ohos alonate kinase 2.7.4.3 aden late kinase 2.7.4.4 nucleoside- ohos hate kinase 6 nucleoside-di hos hate kinase 2.7.4.7 ohos .hometh lo rimidine kinase 2.7.4.8 _uan late kinase 2.7.4.9 dTMP kinase 2.7.4.10 nucleoside-tri ohos aden late kinase 11 deox aden late kinase 2.7.4.12 T2-induced deox nucleotide kinase 2.7.4.13 deox nucleoside-hos hate kinase 14 c id late kinase 2.7.4.15 thiamine-di ohos hate kinase W0 2013/040383 61 PCT/U82012/055469 Enz me Commission No. En me Name 2.7.4.16 thiamine- ohos ohate kinase 3-phosphoglyceroyl—phosphate—polyphosphate 2.7.4.17 o hos o hotransferase 2.7.4.18 farnes l-di ohos hate kinase 2.7.4.19 5-meth ldeox c idine-5'-ohos ohate kinase yl-diphosphate—polyphosphate 2.7.4.20 o hos o sferase 21 ol-hexakis ohos hate kinase 2.7.4.22 UMP kinase 2.7.4.23 ribose 1,5-biSoohoshate okinase 2.7.4.24 di 0 hos o hoinositol- o entakis o hos 0 hate kinase 2.7.4.- Farnes lmonOHhoshate kinase - Geran l-_eran l mono ohos hate kinase 274- Ph 1- oohoshate kinase Phosphomevalonate kinase enzymes are of particular interest.

Phosphomevalonate kinase (EC 2.7.4.2) catalyzes the phosphorylation of phosphomevalonate. This enzyme is encoded by erg8 in Saccharomyces cerevisiae (Tsay et al., Mol. Cell Biol. 11:620-631 (1991)) and mvaK2 in Streptococcus pneumoniae, Staphylococcus aureus and Enterococcusfaecalis (Doun et al., Protein Sci. 14: 1 134-1 139 (2005); Wilding et al., J Bacteriol. 182:4319-4327 (2000)). The Streptococcus pneumoniae and Enterococcusfaecalis enzymes were cloned and characterized in E. coli (Pilloff et a1., JBiol. Chem. 278:4510-4515 (2003); Doun et a1., Protein Sci. 14: 1 134-1 139 (2005)). The S. pneumoniae phosphomevalonate kinase was active on several alternate substrates including cylopropylmevalonate ate, vinylmevalonate phosphate and lmevalonate phosphate (Kudoh et al, Bioorg Med Chem 18:1124-34 (2010)).

GenBank ID GI Number AAA34596.1 171479 19196024261 9937366 AAGoz4s7.1 9937409 19196024423 8

[00143] Farnesyl monophosphate kinase enzymes catalyze the CTP dependent orylation of yl monophosphate t0 farnesyl diphosphate. Similarly, geranylgeranyl phosphate kinase catalyzes CTP dependent phosphorylation. Enzymes with these activities were identified in the microsomal fraction of cultured Nicotiana tabacum W0 2013/040383 62 PCT/U82012/055469 (Thai et al, PNAS 96: 13080-5 (1999)). However, the associated genes have not been fied to date. phate lyase [Figure 1: Step E] Diphosphate lyase enzymes catalyze the conversion of alkyl diphosphates to alkenes. Carbon-oxygen lyases that operate on phosphates are found in the EC 4.2.3 enzyme class. The table below lists several useful enzymes in EC class 4.2.3. Exemplary enzyme candidates were bed above (see phosphate lyase section). 4.2.35 4215 EXAMPLE 11 Preparation of an Isobutylene Producing ial Organism ] This example describes the generation of a microbial organism capable of producing isobutylene from isobutanol, in an organism engineered to have an isobutylene pathway.

An anol-overproducing strain erichia coli is used as a target organism to engineer an isobutylene-producing pathway. Pathways for efficiently converting central metabolic intermediates to isobutanol are known in the art (for example: US 8017375; ; ; ; Dickinson et al., JBC 273 :2575 1-56 (1998)) and isobutanol overproducing E. coli strains have been developed (for e, Atsumi et al, Appl Microbiol Biotech 85:651-57 (2010)).

To generate an E. coli strain engineered to produce isobutylene from anol, nucleic acids encoding the enzymes utilized in the pathway of Figure 1 are expressed in E. coli using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra, 1989). In particular, the mvk (NP_357932.1), mvaK2 457.1) and, z'spS (CAC35696.1 ) genes encoding W0 2013/040383 63 alkyl phosphate kinase, alkyl phate kinase and isobutylene synthetase, respectively, are cloned into the pZEl3 vector (Expressys, Ruelzheim, Germany), under the control of the cO promoter. This plasmid is then transformed into a host strain containing lacIQ, which allows inducible expression by addition of isopropyl-beta-D-l- thiogalactopyranoside (IPTG).

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, ok et al., supra, 2001). The expression of isobutylene pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification ofmRNA and immunoblotting.

Enzymatic activities of the expressed enzymes are ed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce isobutylene is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

[00149] Microbial strains engineered to have a filnctional isobutylene synthesis pathway are filrther ted by optimization for efficient utilization of the pathway.

Briefly, the engineered strain is assessed to determine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, introduction of additional gene copy numbers. Strategies are also applied to improve production of isobutylene sor isobutanoyl-phosphate, such as mutagenesis, g and/or deletion of native genes involved in byproduct formation.

To generate better producers, lic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that onally ze utilization of the pathway (see, for example, US. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in US. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more nt tion of isobutylene. One modeling method is the bilevel optimization ch, OptKnock (Burgard et al., hnol.

Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of ylene. Adaptive evolution also can be used to generate better producers of, for example, the anoyl-phosphate intermediate W0 2013/040383 64 PCT/U82012/055469 or the ylene product. Adaptive evolution is performed to e both growth and production teristics (Fong and Palsson, Nat. Genet. 36:1056-1058 ; Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of ng, genetic engineering and adaptive evolution can be applied to the isobutylene producer to further increase production.

For large-scale production of isobutylene, the above isobutylene y- containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are med in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then g culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). erobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable ls, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX- 087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for c acids, Lin et al., Biotechnol. Bioeng, 9 (2005).

EXAMPLE III Pathways for the formation of butadiene precursor 3-butenol (butenol) from pyruvate and acetaldehyde.

This example describes pathways for converting pyruvate and acetaldehyde to 3-buten-l-ol, and fiarther to butadiene. The conversion of pyruvate and acetaldehyde to 3- buten-l-ol is accomplished in four enzymatic steps. Pyruvate and acetaldehyde are first condensed to 4-hydroxyoxovalerate by 4-hydroxyketovalerate aldolase. The 4- hydroxyoxovalerate product is subsequently dehydrated to 2-oxopentenoate.

Decarboxylation of 2-oxopentenoate yields 3-buten-l-al, which is further reduced to 3- butenol by an alcohol dehydrogenase. s and gene candidates for catalyzing buten-l-ol pathway reactions are described in further detail below.

The condensation of pyruvate and acetaldehyde to 4-hydroxyoxovalerate is zed by 4-hydroxyoxovalerate aldolase (EC 39). This enzyme participates in pathways for the degradation of phenols, cresols and ols. The E. coli enzyme, d by mhpE, is highly specific for acetaldehyde as an acceptor but accepts the alternate substrates 2-ketobutyrate or phenylpyruvate as donors (Pollard et al., Appl Environ Microbiol 64:4093-4094 (1998)). r enzymes are encoded by the cth and todH genes ofPseudomonas putida (Lau et al., Gene 146:7-13 (1994); Eaton, J Bacteriol. 178: 135 1-1362 (1996)). In Pseudomonas CF600, this enzyme is part of a bifunctional aldolase-dehydrogenase heterodimer encoded by dmpFG (Manjasetty et al., Acta Crystallogr.D.Biol Crystallogr. 5 7:582-585 ). The dehydrogenase functionality interconverts acetaldehyde and acetyl-CoA, providing the advantage of reduced cellular concentrations of acetaldehyde, toxic to some cells.

GenBank ID GI Number AAC73455.1 8 Escherichia coli AAB62295.1 1263190 AAA61944.1 485740 CAA43227.1 45684 Pseudomonas s. CF600 26.1 45683 monas SI. CF600 Dehydration of 4-hydroxyoxovalerate to 2-oxopentenoate is catalyzed by 4- hydroxyoxovalerate hydratase (EC 80). This enzyme participates in aromatic degradation pathways and is typically co-transcribed with a gene encoding an enzyme with 4-hydroxyoxovalerate aldolase activity. ary gene products are encoded by mhpD of E. coli (Ferrandez et al., J Bacteriol. 73-2581 (1997); Pollard et al., Eur J Biochem. 251 :98-106 (1998)), todG and cth of Pseudomonas putida (Lau et al., Gene 146:7-13 ; Eaton, riol. 178: 135 1-1362 (1996)), cnbE of nas sp.

CNB-l (Ma et al., Appl Environ Microbiol 73:4477-4483 (2007)) and mhpD of Burkholderia xenovorans (Wang et al., FEBS J 272:966-974 (2005)). A closely related enzyme, 2-oxoheptaene-1,7-dioate hydratase, participates in 4-hydroxyphenylacetic acid degradation, where it converts 2-oxo-heptene-1,7-dioate (OHED) to 2-oxo hydroxy-hepta-1,7-dioate using magnesium as a cofactor (Burks et al., J.Am.Chem.Soc. 120: (1998)). OHED hydratase enzyme candidates have been identified and characterized in E. coli C (Roper et al., Gene 156:47-51 (1995); Izumi et al., JMol.Biol. 370:899-911 (2007)) and E. coli W(Prieto et al., J Bacteriol. 178: 1 1 1-120 (1996)). Sequence comparison reveals homologs in a wide range of bacteria, plants and animals. Enzymes W0 2013/040383 66 with highly similar sequences are contained in ella pneumonia (91% identity, eval = 2e-138) and Salmonella enterica (91% identity, eval = 4e-138), among others.

Protein todG cnbE —_Comamonas So. CNB-l mhD Q13VUO 8582 Barkholderia rans hIcG CAA57202.1 —556840 Escherichia coli C hIaH CAA86044.1 —757830 —Escherichia coli W hIaH ABR80130.1 —150958100 _—Klebsiella Ineamoniae Sari 01896 ABX21779.1 —160865156—Salmonella enterica Decarboxylation of 4-hydroxyoxovalerate is catalyzed by a keto-acid decarboxylase. Suitable enzyme ates include pyruvate decarboxylase (EC 4.1.1.1), benzoylformate decarboxylase (EC 4.1.1.7), alpha-ketoglutarate decarboxylase and branched-chain alpha-ketoacid decarboxylase. Pyruvate decarboxylase (PDC), also termed keto-acid decarboxylase, is a key enzyme in alcoholic fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde. The enzyme from Saccharomyces cerevisiae has a broad substrate range for aliphatic 2-keto acids including 2-ketobutyrate, 2- ketovalerate, 3-hydroxypyruvate and 2-phenylpyruvate (22). This enzyme has been extensively studied, engineered for altered activity, and functionally sed in E. coli (Killenberg-Jabs et al., EarJBiochem. 268:1698-1704 (2001); Li et al., Biochemistry. 38:10004-10012 (1999); ter Schure et al., Appl.Environ.Microbiol. 64:1303-1307 (1998)).

The PDC from Zymomonas mobilas, d by pdc, also has a broad substrate range and has been a subject of ed engineering studies to alter the affinity for different substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The crystal ure of this enzyme is ble (Killenberg-Jabs et al., Ear.J.Biochem. 268:1698-1704 (2001)).

Other well-characterized PDC candidates include the s from Acetobacter pasteurians (Chandra et al., 176:443-451 (2001)) and Klayveromyces lactis (Krieger et al., 269:3256-3263 (2002)).

Protein GenBank ID GI Number dc 1 118391 dIcI P06169 30923172 Saccharom ces cerevisiae dIc Q8L388 20385191 Acetobacter Iastearians dIcI Q12629 52788279 Kla verom ces lactis Like PDC, lformate oxylase (EC 4.1.1.7) has a broad substrate range and has been the target of enzyme engineering studies. The enzyme from Pseudomonas putida has been extensively studied and crystal structures of this enzyme are available (Polovnikova et al., 42:1820-1830 (2003); Hasson et al., 37:9918-9930 (1998)).

Site-directed mutagenesis of two residues in the active site of the Pseudomonas putida enzyme altered the affinity (Km) of naturally and non-naturally occurring substrates (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)). The properties of this enzyme have been further modified by directed engineering (Lingen et al., Chembiochem. 4:721- 726 (2003); Lingen et al., Protein Eng -593 ). The enzyme from monas aeruginosa, encoded by mdlC, has also been characterized experimentally (Barrowman et al., 34:57-60 (1986)). Additional gene candidates from Pseudomonas stutzeri, Pseudomonasfluorescens and other organisms can be inferred by sequence homology or identified using a growth selection system developed in Pseudomonas putida (Henning et al., Appl.Environ.Microbiol. 72:7510-7517 (2006)). k ID GI Number P209062 3915757 Q9HUR2.1 81539678 II-ABN804231 126202187 _YP260581.1 70730840 A third enzyme capable of decarboxylating 2-oxoacids is alpha-ketoglutarate decarboxylase (KGD). The substrate range of this class of enzymes has not been studied to date. An exemplarly KDC is encoded by kad in Mycobacterium tuberculosis (Tian et al., PNAS 102: 10670-10675 (2005)). KDC enzyme activity has also been detected in several species of rhizobia including Bradyrhizobiumjaponicum and Mesorhizobium loti (Green et al., J Bacteriol 182:2838-2844 (2000)). Although the KDC-encoding gene(s) have not been isolated in these sms, the genome sequences are available and several genes in each genome are annotated as putative KDCs. A KDC from Euglena gracilis has also been characterized but the gene associated with this activity has not been identified to date (Shigeoka et al., iochem.Biophys. 288:22-28 (1991)). The first twenty amino acids starting from the inus were sequenced VKDVKFLLDKVFKV (SEQ ID NO. ) (Shigeoka and , Arch.Biochem.Biophys. 288:22-28 (1991)). The gene could W0 2013/040383 68 be identified by testing candidate genes containing this N—terminal sequence for KDC activity.

GenBank ID GI Number 0504634 160395583 M cobacterium ulosis NP 7670921 27375563 Brad rhizobium 'aonicum USDAIIO 204.1 13473636 Mesorhizobium loti A fourth candidate enzyme for catalyzing this reaction is branched chain alpha- ketoacid oxylase (BCKA). This class of enzyme has been shown to act on a variety of nds varying in chain length from 3 to 6 carbons (Oku et al., JBiol Chem. 263 : 1 8386-18396 (1988); Smit et al., Appl Environ Microbiol 71:303-311 ). The enzyme in Lactococcus lactis has been characterized on a variety of branched and linear substrates including 2-oxobutanoate, 2-oxohexanoate, entanoate, 3-methyl oxobutanoate, 4-methyloxobutanoate and isocaproate (Smit et al., Appl n iol 71 :303-311 (2005)). The enzyme has been structurally characterized (Berg et al., Science. 318:1782-1786 (2007)). ce alignments n the occus lactis enzyme and the pyruvate decarboxylase ofZymomonas mobilus indicate that the catalytic and substrate recognition residues are nearly identical (Siegert et al., Protein Eng Des Sel 18:345-357 (2005)), so this enzyme would be a promising candidate for directed engineering. Decarboxylation of alpha-ketoglutarate by a BCKA was detected in Bacillus subtilis; r, this activity was low (5%) relative to activity on other branched-chain substrates (Oku and Kaneda, J Biol Chem. 386-18396 ) and the gene encoding this enzyme has not been identified to date. Additional BCKA gene candidates can be identified by homology to the Lactococcus lactis protein sequence. Many of the high-scoring BLASTp hits to this enzyme are annotated as indolepyruvate decarboxylases (EC 4.1.1.74). Indolepyruvate decarboxylase (IPDA) is an enzyme that catalyzes the decarboxylation of indolepyruvate to indoleacetaldehyde in plants and plant bacteria.

Recombinant branched chain alpha-keto acid decarboxylase enzymes derived from the E1 ts of the mitochondrial branched-chain keto acid dehydrogenase complex from Homo sapiens and Bos taurus have been cloned and functionally expressed in E. coli (Davie et al., J.Biol. Chem. 267: 16601-16606 (1992); Wynn et al., J.Biol. Chem. 267: 12400-12403 (1992); Wynn et al., J.Biol. Chem. 267: 1881-1887 (1992)). In these studies, the authors found that co-expression of chaperonins GroEL and GroES enhanced the specific activity of the decarboxylase by 500-fold (Wynn et al., J.Biol. Chem.

W0 2013/040383 69 267: 12403 (1992)). These s are ed of two alpha and two beta subunits .

Protein GenBank ID GI Number deA AAS49166.1 44921617 Lactococcus lactis BCKDHB NP 898871.1 34101272 Homo satiens BCKDHA NP 0007001 11386135 Homo satiens BCKDHB P21839 1 15502434 Bos taurus BCKDHA P11178 129030 Bos taurus Reduction of 3-butenal to nol is catalyzed by an aldehyde reductase or alcohol dehydrogenase. Genes encoding s that catalyze the reduction of an aldehyde to alcohol (i.e., alcohol dehydrogenase or equivalently aldehyde reductase) include aer encoding a medium-chain alcohol dehydrogenase for C2-C14 (Tani et al., Appl.Environ.Microbiol. 66:5231-5235 (2000)), thD andfucO from E. coli (Sulzenbacher et al., 342:489-502 ), and bdh I and bdh II from C. acetobutylz'cum which converts butyryaldehyde into l (Walter et al., 174:7149-7158 (1992)). Yth catalyzes the reduction of a wide range of aldehydes using NADPH as the cofactor, with a preference for chain lengths longer than C(3) (Sulzenbacher et al., 342:489-502 (2004);Perez et al., J Biol. Chem. 283:7346-7353 (2008)). The adhA gene product from Zymomonas mobilisE has been demonstrated to have activity on a number of aldehydes including formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and acrolein (Kinoshita et al., Appl Microbiol Biotechnol 22:249-254 (1985)). Additional aldehyde reductase candidates are encoded by bdh in C. saccharoperbutylacetom'cum and Cbez'_1 722, Cbez’_2181 and 2421 in C. Beijerz’nckz’z’. Additional aldehyde reductase gene candidates in Saccharomyces cerevisiae include the de reductases GRE3, ALD2-6 and HFDl, glyoxylate reductases GORl and YPL113C and glycerol dehydrogenase GCYl (A1; Atsumi et al., Nature 451 :86-89 (2008)).

The enzyme candidates described usly for catalyzing the reduction of methylglyoxal to acetol or lactaldehyde are also suitable lactaldehyde ase enzyme candidates.

NP 4172791 16130706 ichia coli 19th NP 3498921 15896543 Clostrz'dz'um acetobu licum bdh 11 NP .1 15896542 Clostrz'dz'um acetobu licum Clostrz'dz'um saccharmerbu m'cum Clostrz'dz'um bez’ 'erinckii Clostrz'dz'um bez’ 'erz’nckz'z' z'dz'um bez’ 'erz’nckz'z' Saccharom ces cerevisiae Saccharom ces cerevisiae Saccharom ces cerevisiae Saccharom ces siae rom ces cerevisiae Saccharom ces cerevisiae Saccharom ces cerevisiae GORI NP 1 6324055 Saccharom ces cerevisiae YPLI13C AAB68248.1 1163100 Saccharom ces cerevisiae GCYI CAA99318.1 1420317 Saccharom ces cerevisiae s exhibiting 4-hydroxybutyrate dehydrogenase activity (EC 1.1.1.61) and glutarate semialdehyde reductase also fall into this ry. 4-Hydroxybutyrate dehydrogenase s have been characterized in Ralstom'a eutropha (Bravo et al., J Forens Sci, 49379-3 87 (2004)) and Clostridium kluyverz' (Wolff et al., Protein Expr.Purl'f. 6:206-212 (1995)). Yet another gene is the alcohol ogenase adhl from Geobacz’llus thermoglucosidasius (Jeon et al., JBiotechnol 135127-133 (2008)). Glutarate semialdehyde ase enzymes include the ATEG_00539 gene product ofAspergillus terreus and 4-hydroxybutyrate dehydrogenase ofArabidopsis thaliana, encoded by 4hbd (A2). The A. thaliana enzyme was cloned and characterized in yeast (Breitkreuz et al., J.Bl'0[. Chem. 278:41552-41556 (2003)).

PROTEIN GENBANK ID GI NUMBER ORGANISM 4hbd YP 7260531 113867564 Ralstom'a eutrOIha H16 4hbd L21902.1 146348486 Clostrz'dz'um klu verz' DSM 555 AAR91477.1 40795502 Geobacillus thermo lucosz'dasz'us ATEG 00539 XP 0012106251 115491995 ASIer illus terreus NIH2624 4hbd AAK94781.1 15375068 OIsz's thaliana ] Another exemplary aldehyde reductase is methylmalonate semialdehyde reductase, also known as 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). This enzyme participates in , leucine and cine degradation and has been identified in bacteria, eukaryotes, and mammals. The enzyme encoded by P84067 from Thermus thermophilus HB8 has been structurally characterized (Lokanath et al., JM01 Biol, 352:905-17 (2005)). The reversibility of the human 3-hydroxyisobutyrate dehydrogenase was demonstrated using isotopically-labeled substrate (Manning et al., Biochem J, 231 :481-4 (1985)). onal genes encoding this enzyme include 3hz'dh in Homo sapiens (Hawes et al., Methods Enzymol, 324:218-228 (2000)) and Oryctolagus cum’culus (Hawes et a1., supra; Chowdhury et a1., Bioscz'.Bz'otechnol Biochem. 60:2043-2047 (1996)), mmsB in Pseudomonas aeruginosa and monas putida, and dhat in Pseudomonas putida (Aberhart et al., J Chem.Soc.[Perkin I] 6:1404-1406 (1979); Chowdhury et al., Bioscz'.Bz'otechnol Biochem. 60:2043-2047 (1996); Chowdhury et a1., Bioscz'.Bz'otechnol Biochem. 67:43 8-441 (2003)). Several 3-hydroxyisobutyrate dehydrogenase enzymes have been characterized in the reductive direction, including mmsB from Pseudomonas aeruginosa (Gokam et al., US Patent 739676, (2008)) and mmsB from Pseudomonas putida.

PROTEIN GENBANK ID GI NUMBER ORGANISM P84067 P84067 75345323 s thermOIhz'lus 3hz'dh P31937.2 12643395 Homo saIl'ens 3hz'dh 0r ctolaus ulus mmsB Pseudomonas Iutz'da mmsB monas aeru inosa dhat Pseudomonas Iutz'da e IV Preparation of a ene Producing Microbial Organism with a Butenol Pathway This example describes the generation of a ial organism capable of producing butadiene from pyruvate via a butenol intermediate, in an organism engineered to have a butadiene pathway.

[00164] ichia coli is used as a target sm to engineer a butadiene-producing pathway. E. 6011' provides a good host for generating a non-naturally occurring microorganism capable of producing butadiene. E. coli is amenable to genetic manipulation and is known to be e of producing various products, including ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce the butadiene pathway precursor, butenol, a functional nucleic acids encoding the enzymes utilized in the pathway described in Example 111, are expressed in E. 6012' using well known molecular biology techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra, 1989).

In particular, an E. coli strain is ered to produce butenol from te via the route described in Example 3. For the first stage of pathway construction, genes encoding enzymes to transform pyruvate to butenol are assembled onto a vector. The genes mhpE (AAC73455.1), mhpD (AAC734532), kch (AAS49166.1), adhA (YP_162971.1) encoding 4-hydroxyoxovalerate aldolase, 4-hydroxy oxovalerate dehydratase, 2-oxopentenoate decarboxylase and 3-butenal reductase, respectively, are cloned into the pZE13 vector (Expressys, Ruelzheim, Germany), under the control of the PAl/lacO promoter. The genes mvk (NP_357932.1), mvaK2 (AAG02457.1) and, ispS (CAC35696.1) ng alkyl phosphate , alkyl phate kinase and butadiene synthetase, tively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the cO promoter. The two plasmids are transformed into E. 6012' host strain containing lacIQ, which allows inducible expression by on of isopropyl-beta-D-l-thiogalactopyranoside (IPTG).

The resulting genetically engineered organism is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The sion of butadiene pathway genes is corroborated using methods well known in the art for determining polypeptide expression or enzymatic activity, including for example, Northern blots, PCR amplification ofmRNA and immunoblotting.

Enzymatic activities of the expressed enzymes are ed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce butadiene is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a filnctional butadiene synthesis pathway are filrther augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is ed to ine whether any of the exogenous genes are expressed at a rate limiting level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for e, introduction of additional gene copy s. Strategies are also applied to improve production of 73 2012/055469 butadiene precursor en-l-ol, such as mutagenesis, cloning and/or deletion of native genes ed in uct formation.

To generate better butadiene producers, metabolic modeling is utilized to optimize growth conditions. Modeling is also used to design gene knockouts that additionally optimize ation of the y (see, for example, US. patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in US. Patent No. 7,127,379). Modeling analysis allows reliable predictions of the effects on cell growth of shifting the metabolism towards more efficient production of butadiene. One modeling method is the l optimization approach, OptKnock (Burgard et al., Biotechnol.

Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of butadiene. Adaptive evolution also can be used to generate better producers of, for example, the buten-l-ol ediate or the butadiene t. Adaptive evolution is performed to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of modeling, genetic ering and adaptive evolution can be applied to the butadiene producer to further se tion.

For large-scale production of butadiene, the above butadiene pathway- ning organism is cultured in a fermenter using a medium known in the art to t growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous manner. Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2SO4. The growth rate is determined by measuring optical density using a spectrophotometer (600 nm), and the glucose uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable alcohols, organic acids, and residual glucose can be quantified by HPLC (Shimadzu) with an HPX- 087 column (BioRad), using a refractive index detector for glucose and alcohols, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng, 775-779 (2005).

EXAMPLE V W0 20131040383 74 2012/055469 Pathway for the formation of butadiene precursor 3-butenol (butenol) from acrylyl-CoA This example describes pathways for converting acryly-CoA to nol, and fithher to butadiene. The conversion of acrylyl-CoA to 3-butenol is accomplished in four enzymatic steps. Acrylyl-CoA and acetyl-CoA are first condensed to 3-0X0pent enoyl-CoA by a beta-ketothiolase. The 3-0X0pentenoyl-C0A product is subsequently hydrolyzed to 3-oxopentenoate by a CoA hydrolase, transferase or synthetase.

Decarboxylation of the acid intermediate yields 3-butenal, which is r reduced to 3-butenol by an alcohol dehydrogenase or ketone reductase.

[00172] Enzymes and gene candidates for catalyzing butenol pathway reactions are described in further detail below.

Acrylyl-CoA and acetyl-CoA are condensed to form 3-0X0pentenoyl-C0A by a beta-ketothiolase (EC 2.3.1.16). etothiolase enzymes catalyzing the formation of beta-ketovalerate from acetyl-CoA and propionyl-COA are good candidates for catalyzing the formation of 3-oxopenenoyl-COA. Z00gloea ramigera possesses two ketothiolases that can form beta-ketovaleryl-COA from propionyl-COA and acetyl-CoA and R. eutropha has a beta-oxidation ketothiolase that is also e of catalyzing this transformation (Gruys et al., US. Patent No. 5,958,745). The sequences of these genes or their translated proteins have not been reported, but several genes in R. eutropha, Z. ramigera, or other sms can be identified based on sequence homology to bkt3 from R. 611110191111.

Protein 0r-anism I11aA Ralstom'a 6111101161 1116 A1 713 YP 7262051 113867716 Ralstom'a 6111101161 IcaF Ralstom'a 6111101161 1116 31369 m'a eutrOI11a 1116 A0170 YP 1 201 Ralstom'a 6111101161 1116 A0462 m'a eutrOI11a 1116 A1528 YP 7260281 113867539 Ralstom'a eutrOI11a 1116 30381 YP 1 116694334 Ralstom'a eutrOI11a 1116 30662 Ralstom'a eutrOI11a 1116 30759 YP 728921.1 116694710 Ralstom'a eutrOI11a 1116 30668 YP 7288301 116694619 Ralstom'a eutrOI11a 1116 A1720 YP 7262121 113867723 Ralstom'a 6111101161 1116 A1887 YP 7263561 113867867 Ralstom'a 11a I11bA Z00 106a ramz' era bk13 CuIriavidus taiwanensz's W0 2013/040383 75 Rmet I362 YP 5835141 94310304 nia metallidurans BIh 0975 YP 0018572101 186475740 lderia Ih matum Additional enzymes include beta-ketothiolases that are known to convert two molecules of acetyl-CoA into acetoacetyl-CoA (EC 2.1.3.9). Exemplary acetoacetyl-CoA thiolase s include the gene products of atoB from E. coli (Martin et al., Nat.

Biotechnol. 21 :796-802 (2003)), thlA and thlB from C. acetobutylicum (Hanai et al., Appl.

Environ. Microbiol. 73:7814-7818 (2007); Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541 (2000)), and ERGIO from S. cerevisiae (Hiser et al., J. Biol. Chem. 269:31383- 31389 (1994)).

Protein k ID GI Number atoB NP 416728 16130161 Escherichia coli thlA NP 3494761 15896127 Clostriclium acetobu licum thlB NP 1492421 15004782 Clostriclium acetobu licum ERGIO NP 015297 6325229 Saccharom ces cerevisiae

[00175] Beta-ketoadipyl-CoA thiolase (EC 2.3.1.174), also called 3-oxoadipyl-CoA thiolase, converts beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA, and is a key enzyme of the beta-ketoadipate pathway for aromatic compound degradation. The enzyme is widespread in soil bacteria and fungi including Pseudomonas putida (Harwood et al., J.

Bacteriol. 1766488 (1994)) and Acinetobacter calcoaceticus (Doten et al., J.

Bacteriol. 169:3168-3174 (1987)). The P. puticla enzyme is a tramer g 45% sequence homology to beta-ketothiolases involved in PHB synthesis in nia eutropha, fatty acid degradation by human mitochondria and butyrate production by Clostriclium acetobulylicum (Harwood et al., supra). A beta-ketoadipyl-CoA thiolase in monas knackmussii (formerly sp. 313) has also been characterized (Gobel et al., J.

Bacteriol. 184:216-223 (2002); Kaschabek et al., supra).

GenBank ID GI Number NP 1 506695 Pseudomonas Iaticla IcaF AAC37148.1 141777 Acinetobacter calcoaceticus _catF Q8VPF1.1 754045 81 Pseudomonas knackmussii Removal of the CoA moiety of 3-oxopentenoyl-CoA product is catalyzed, for e, by 3-oxopentenoyl-CoA ase. The CoA hydrolase encoded by acot12 from Rattus norvegicus brain (Robinson et al., Biochem.Biophys.Res.Comman.

WO 40383 76 71 :95 9-965 (1976)) can react with several alternate ates including butyryl-CoA, hexanoyl-CoA and malonyl-CoA. The human dicarboxylic acid thioesterase, encoded by acot8, exhibits activity on glutaryl-CoA, adipyl-CoA, suberyl-CoA, sebacyl-CoA, and dodecanedioyl-CoA (Westin et al., J.Biol. Chem. 280:38125-38132 (2005)). The closest E. coli homolog to this enzyme, tesB, can also hydrolyze a range of CoA thiolesters (Naggert et al., JBiol Chem 266: 1 1044-1 1050 (1991)). A similar enzyme has also been characterized in the rat liver (Deana R., Biochem Int 26:767-773 (1992)). Additional enzymes with hydrolase activity in E. coli include ybgC, paaI, and )2de tsova, et al., FEMS Microbiol Rev, 2005, 29(2):263-279; Song et al., JBiol Chem, 2006, 281(16): 1 1028-3 8). Though its sequence has not been reported, the enzyme from the mitochondrion of the pea leaf has a broad substrate city, with demonstrated activity on -CoA, propionyl-CoA, butyryl-CoA, palmitoyl-CoA, oleoyl-CoA, succinyl-CoA, and crotonyl-CoA r et al., Plant.Physiol. 94:20-27 (1990)) The acetyl-CoA hydrolase, ACHI , from S. cerevisiae represents another candidate hydrolase (Buu et al., 1.5701. Chem. 278:17203-17209 (2003)) .

GenBank Accession # GI# Or_anism \P 5701031 18543355 Rattusnorveicus \P 414986 16128437 Escherichia coli CAA15502 0 Homo satiens 69 Rattus norve icus 16128580 Escherichia coli P 009538 6319456 Saccharom ces cerevisiae onal hydrolase enzymes include oxyisobutyryl-CoA hydrolase which has been described to efficiently catalyze the sion of oxyisobutyryl- CoA to 3-hydroxyisobutyrate during valine degradation (Shimomura et al., JBiol Chem. 269:14248-14253 (1994)). Genes encoding this enzyme include hibch ofRattus icus (Shimomura et al., Methods Enzymol. 324:229-240 (2000)) and Homo sapiens (Shimomura et al., supra). Similar gene candidates can also be identified by sequence homology, including hibch of Saccharomyces cerevisiae and BC_2292 of Bacillus cereus.

GenBank Accession # Q5XIE6-2 146324906 hibch Q6NVY1.2 146324905 W0 2013/040383 77 Protein GenBank Accession # hibch P28817.2 2506374 Saccharom ces cerevisiae BC 2292 AP09256 29895975 Bacillus cereus Decarboxylation of 3-oxopentenoate is catalyzed by a 3-ketoacid decarboxylase. The acetoacetate decarboxylase (EC 4) from Clostriclium acetobutylicum, encoded by adc, has a broad substrate specificity and has been shown to decarboxylate numerous alternate substrates including 2-ketocyclohexane carboxylate, 3- oxopentanoate, 2-oxophenylpropionic acid, 2-methyloxobutyrate and benzoyl- acetate (Rozzel et al., J.Am.Chem.S0c. 106:4937-4941 ; Benner and Rozzell, J.Am.Chem.S0c. [03:993-994 ; Autor et al., JBi0l. Chem. 245:5214-5222 (1970)).

An acetoacetate decarboxylase has also been characterized in Clostriclium beijerinckii (Ravagnani et al., MolMicrobiol 37: 1 172-1 185 (2000)). The acetoacetate decarboxylase from us polymyxa, characterized in cell-free extracts, also has a broad substrate specificity for 3-keto acids and can decarboxylate 3-oxopentanoate (Matiasek et al., Curr.Micr0bi0l 42:276-281 (2001)). The gene encoding this enzyme has not been identified to date and the genome sequence of B. polymyxa is not yet available. Another adc is found in Clostridium roperbutylacetonicum (Kosaka, et al., Bi0sci.Bi0teclm0l Biochem. 71 :5 8-68 (2007)). onal gene candidates in other organisms, including Clostriclium num and us amyloliquefaciens FZB42, can be identified by sequence homology.

GenBank ID NP 1493281 15004868 Clostridium acetobu licum adc AAP42566.1 31075386 Clostridium saccharo I erbu lacetonicum YP 0013109061 150018652 Clostridium bei'erinckii CLL A2135 YP 0018863241 187933144 Clostridium botulinum RBAM 030030 YP 0014225651 154687404 Bacillus am l0lilue aciens

[00179] Reduction of 3-butenal to 3-butenol is catalyzed by an l dehydrogenase or ketone reductase. l dehydrogenases described above in Example 111 are also suitable candidates for this transformation. There exist several exemplary l dehydrogenases that convert a ketone to a yl functional group. Two such enzymes from E. c0li are encoded by malate dehydrogenase (mdh) and lactate ogenase (lclhA). In addition, lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high ties on 2-ketoacids of various chain lengths includings e, 2-oxobutyrate, entanoate and 2-oxoglutarate (Steinbuchel et al., EurJBiochem. 130:329-334 (1983)). Conversion of alpha-ketoadipate into alpha- yadipate can be catalyzed by 2-ketoadipate reductase, an enzyme reported to be found in rat and in human placenta (Suda et al., Arch.Biochem.Bi0phys. 176:610-620 (1976); Suda et al., Biochem.Bi0phys.Res.Comman. 77:586-591 (1977)). An additional oxidoreductase is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from the human heart which has been cloned and terized (Marks et al., J.Biol. Chem. 267: 15459-15463 (1992)). Alcohol dehydrogenase enzymes of C. beijerinckii (Ismaiel et al., JBacteriol. 97-5105 (1993)) and T. brockii (Lamed eta1.,Biochem.J. 195:183- 190 (1981); Peretz et al., Biochemistry. 28:6549-6555 (1989)) convert acetone to isopropanol. Methyl ethyl ketone reductase catalyzes the reduction ofMEK to 2-butanol.

Exemplary MEK reductase enzymes can be found in Rhodococcus ruber (Kosjek et al., Biotechnol Bioeng. 86:55-62 (2004)) and Pyrococcasfuriosus (van der a1., EurJBiochem. 268:3062-3068 (2001)). ——_— ——_— ——__— 352.1 177198 B593 HTD4 —CAD36475 21615553 Rhodococcus ruber —AAC25556 3288810 Enzymes that catalyze the reduction of 3-oxobutanol to 1,3-butanediol are also applicable here. Such enzymes are found in organisms of the genus Bacillus, acterium, Candida, and Klebsiella among others, as described by Matsuyama et al.

JM01 Cat B Enz, 11:513-521 (2001). One of these enzymes, SADH from Candida parapsilosis, was cloned and characterized in E. coli. A mutated Rhodococcus phenylacetaldehyde ase (Sar268) and a Leifonia alcohol ogenase have also been shown to catalyze this transformation at high yields (Itoh et al., Appl.Micr0bi0[ Biotechnol. 75:1249-1256 (2007)).

GenBank Accession No.

BAA24528.1 2815409 Candida Harasilosis EXAMPLE VI ation of a Butadiene Producing ial Organism with a Butenol Pathway This example bes the generation of a microbial sm capable of producing butadiene from te via a butenol intermediate, in an organism engineered to have a butadiene pathway.

Escherichia coli is used as a target organism to engineer a butadiene-producing pathway. E. 6011' provides a good host for generating a non-naturally occurring microorganism capable of producing butadiene. E. coli is amenable to genetic manipulation and is known to be capable of producing various products, including ethanol, acetic acid, formic acid, lactic acid, and succinic acid, effectively under anaerobic or microaerobic conditions.

] To generate an E. coli strain engineered to e the butadiene pathway precursor, butenol, a functional nucleic acids encoding the enzymes utilized in the pathway described in e 111, are expressed in E. 6012' using well known molecular y techniques (see, for example, Sambrook, supra, 2001; Ausubel supra, 1999; Roberts et al., supra, 1989).

In particular, an E. coli strain is engineered to produce butenol from acrylyl-COA via the route described in Example 111. For the first stage of pathway construction, genes ng enzymes to transform acrylyl-CoA to butenol are assembled onto a vector. The genes phaA (YP_725941.1), tesB 4986), adc (NP_149328.1) and sadh (BAA24528.1) encoding beta-ketothiolase, 3-0x0penten0yl- CoA hydrolase, 3-oxopentenoate oxylase and 3-butenone reductase, respectively, are cloned into the pZEl3 vector (Expressys, Ruelzheim, Germany), under the control of the PAl/lacO promoter. The genes mvk (NP_357932.1), mvaK2 (AAG02457. 1) and, ispS (CAC35696. 1) encoding alkyl phosphate kinase, alkyl diphosphate kinase and butadiene synthetase, respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim, Germany) under the PAl/lacO promoter. The two plasmids are ormed into E. 6012' host strain containing lale, which allows inducible expression by addition of isopropyl-beta-D-l-thiogalactopyranoside (IPTG).

W0 2013/040383 80 The resulting genetically engineered sm is cultured in glucose containing medium following procedures well known in the art (see, for example, Sambrook et al., supra, 2001). The expression of butadiene pathway genes is corroborated using methods well known in the art for determining ptide expression or enzymatic activity, including for example, Northern blots, PCR amplification ofmRNA and immunoblotting.

Enzymatic activities of the expressed enzymes are confirmed using assays specific for the individually activities. The ability of the engineered E. coli strain to produce butadiene is confirmed using HPLC, gas chromatography-mass spectrometry (GCMS) or liquid tography-mass spectrometry (LCMS).

[00186] Microbial strains engineered to have a onal butadiene synthesis pathway are filrther augmented by optimization for efficient utilization of the pathway. Briefly, the engineered strain is assessed to determine r any of the exogenous genes are expressed at a rate ng level. Expression is increased for any enzymes expressed at low levels that can limit the flux through the pathway by, for example, uction of additional gene copy numbers. Strategies are also applied to improve production of butadiene precursor butenol, such as mutagenesis, cloning and/or deletion of native genes involved in byproduct formation.

To te better butadiene producers, metabolic modeling is utilized to ze growth conditions. Modeling is also used to design gene knockouts that additionally optimize utilization of the pathway (see, for example, US. patent ations US 2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466, and in US. Patent No. 7,127,379). Modeling analysis allows le predictions of the effects on cell growth of shifting the metabolism towards more efficient production of butadiene. One modeling method is the bilevel optimization approach, OptKnock (Burgard et al., Biotechnol.

Bioengineer. 84:647-657 (2003)), which is applied to select gene knockouts that collectively result in better production of butadiene. Adaptive evolution also can be used to te better producers of, for example, the butenol intermediate or the butadiene t. Adaptive evolution is med to improve both growth and production characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)). Based on the results, subsequent rounds of ng, genetic engineering and adaptive evolution can be applied to the butadiene producer to further increase production.

W0 2013/040383 81 PCT/U82012/055469 For scale production of butadiene, the above butadiene pathway- containing organism is cultured in a fermenter using a medium known in the art to support growth of the organism under anaerobic conditions. Fermentations are performed in either a batch, fed-batch or continuous . Anaerobic conditions are maintained by first sparging the medium with nitrogen and then sealing culture vessel (e.g., flasks can be sealed with a septum and crimp-cap). Microaerobic conditions also can be utilized by providing a small hole for limited aeration. The pH of the medium is maintained at a pH of 7 by addition of an acid, such as H2804. The growth rate is ined by measuring optical density using a spectrophotometer (600 nm), and the e uptake rate by monitoring carbon source depletion over time. Byproducts such as undesirable ls, organic acids, and residual glucose can be quantified by HPLC dzu) with an HPX- 087 column (BioRad), using a refractive index detector for e and ls, and a UV detector for organic acids, Lin et al., Biotechnol. Bioeng, 775-779 (2005).

Throughout this application various publications have been referenced. The disclosures of these publications in their entireties, including GenBank and GI number publications, are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention.

Claims (13)

What we claim is:

1. A non-naturally occurring microbial organism, said microbial organism having an alkene pathway and comprising at least one exogenous nucleic acid encoding an alkene pathway enzyme expressed in a sufficient amount to convert an alcohol to an alkene, wherein said alkene pathway comprises a pathway selected from: (1) an alcohol kinase and a phosphate lyase; (2) a diphosphokinase and a diphosphate lyase; and (3) an l kinase, an alkyl phosphate kinase and a diphosphate lyase, wherein said microbial organism converts ethanol to ethylene, anol to propylene, isopropanol to propylene, n-butanol to ene, isobutanol to isobutylene, tanol to isobutylene, butanol to butene or butene, pentanol to pent ene, 3-methylbutanol to 3-methylbutene, pentanol to pentene, pentalol to pentene, 2-methylbutanol to 2-methylbutene, 3-methylbutanol to 3- methylbutene, 2-methylbutanol to 2-methylbutene or 2-methylbutene, but- 3-enol to 1,3-butadiene, butenol to 1,3-butadiene, ylethanol to styrene, 2-phenylethanol to styrene, butenol to 1,3-butadiene, 1-octanol to 1-octene, 2- octanol to 2-octene, 3-octanol to ne, or 4-octanol to 4-octene.

2. The non-naturally occurring ial organism of claim 1, wherein said microbial organism comprises two or three exogenous nucleic acids each encoding an alkene pathway .

3. The non-naturally occurring microbial organism of claim 2, wherein said two ous nucleic acids encode an alcohol kinase and a phosphate lyase.

4. The non-naturally ing microbial organism of claim 2, wherein said two exogenous nucleic acids encode a diphosphokinase and a diphosphate lyase.

5. The non-naturally occurring microbial organism of claim 2, wherein said three exogenous nucleic acids encode an alcohol kinase, an alkyl phosphate kinase and a diphosphate lyase.

6. The non-naturally occurring microbial organism of any one of claims 1 to 5, wherein said at least one exogenous nucleic acid is a heterologous nucleic acid.

7. The non-naturally occurring microbial organism of any one of claims 1 to 6, wherein said non-naturally occurring ial organism is in a substantially anaerobic culture medium.

8. A method for ing an alkene comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce said alkene, wherein said alkene is selected from the group consisting of ethylene, propylene, butene, isobutylene, butene, ene, -ene, 3-methylbutene, pent ene, 2-methylbutene, ylbutene, 2-methylbutene, 3-methylbuta-1,2-diene, 1,3- butadiene, 1-octene, 2-octene, 3-octene, 4-octene, and styrene.

9. The method of claim 8, wherein said cell produces 1-octene.

10. The method of claim 8, further comprising separating the ethylene, propylene, butene, isobutylene, butene, butene, pentene, 3-methylbutene, pentene, 2- methylbutene, 2-methylbutene, 2-methylbutene, 3-methylbuta-1,2-diene, 1,3-butadiene, 1-octene, 2-octene, 3-octene, ne, and styrene from the non-naturally ing microorganism.

11. The method of claim 10, wherein said tion ses isolating said alkene from said non-naturally occurring microorganism using distillation, or crystallization.

12. A non-naturally occurring ial organism according to claim 1 substantially as herein described or exemplified.

13. A method ing to claim 8, substantially as herein described or exemplified.