PATENT DOCUMENT

Abstract:
The present disclosure identifies methods and compositions for modifying microbial cells, such that the organisms efficiently synthesize terminal olefins, and in particular the use of such organisms for the commercial production of propylene and related molecules.

Full Description:
SEQUENCE LISTING 
       [0001]    The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 17, 2013, is named “23782US_CRF_sequencelisting.txt”, lists 104 sequences and is 887 KB in size. 
       FIELD OF THE INVENTION 
       [0002]    The present disclosure relates to methods for conferring terminal olefin-producing properties to a heterotrophic or photoautotrophic microbial cell, such that the modified microbial cells can be used in the commercial production of terminal olefins. 
       BACKGROUND OF THE INVENTION 
       [0003]    A terminal olefin is an unsaturated organic compound with a carbon chain backbone, having at least one double bond at the end of the carbon chain. Synthesis of terminal olefins, such as propylene, has significant utility from an industrial prospective. 
         [0004]    Propylene is a terminal olefin molecule of chemical formula C 3 H 6  which is used to manufacture polyethylene, polypropylene, alpha olefins, and styrene. It is also used industrially to produce materials such as polyester, acrylics, ethylene glycol antifreeze, polyvinyl chloride (PVC), propylene oxide, oxo alcohols, and isopropanol. Propylene can be derived from fractional distillation from hydrocarbon mixtures obtained from cracking and other refining processes. However, propylene production by engineered host cells represents a significant alternative to traditional methods of production. 
         [0005]    A need exists therefore, for photosynthetic and non-photosynthetic strains which can make terminal olefins such as propylene and related molecules. 
       SUMMARY OF THE INVENTION 
       [0006]    The disclosure provides a microbial cell for producing a hydrocarbon comprising a recombinant sulfotransferase protein activity and/or a recombinant thioesterase protein activity, wherein the cell synthesizes at least one terminal olefin. The disclosure further provides a method for producing a terminal olefin, comprising culturing an engineered microbial cell in a culture medium, wherein the engineered microbial cell comprises a set of recombinant enzymes comprising at least one sulfotransferase domain and/or at least one thioesterase domain; and isolating the terminal olefin from the microbial cell or the culture medium. In one embodiment of the invention, the microbial cell comprises a nonA gene. In another embodiment, the microbial cell comprises a recombinantly expressed protein comprising any of SEQ ID NOs: 1-3. In an alternative embodiment, the microbial cell comprises a recombinantly expressed protein selected from Tables 1-3 (SEQ ID NOS 4-104, respectively, in order of appearance). 
         [0007]    In one aspect of the invention, the microbial cell is a gram-negative or gram-positive bacterium. In another aspect of the invention, the microbial cell is capable of photosynthesis. In still another aspect, the microbial cell is a cyanobacterium. In yet another aspect, the microbial cell comprises endogenous 3-hydroxybutyryl-ACP and/or endogenous 3-hydroxybutyryl-CoA. 
         [0008]    In one embodiment, the microbial cell is engineered to synthesize 3-hydroxybutyryl-ACP. In another embodiment, the engineering comprises expressing in the microbial cell a recombinant accBCAD gene or a recombinant fabDHG gene. In still another embodiment, the engineering comprises expressing in said microbial cell a genetically modified gene encoding a polypeptide comprising 3-hydroxyacyl-ACP dehydratase activity. In a further embodiment, the engineered microbial cell has a reduced 3-hydroxyacyl-ACP dehydratase activity as compared to a control microbial cell that does not express the genetically modified gene encoding a polypeptide comprising 3-hydroxyacyl-ACP dehydratase activity. In still another embodiment, the genetic modification knocks out an endogenous gene encoding a polypeptide comprising 3-hydroxyacyl-ACP dehydratase activity. In yet another embodiment, the genetically modified gene encoding a polypeptide comprising 3-hydroxyacyl-ACP dehydratase activity is under the control of an inducible promoter. In another embodiment, the microbial cell is cultured in the presence of long chain fatty acids. In one embodiment, the microbial cell produces propylene. 
         [0009]    The invention provides for a microbial cell engineered to synthesize 3-hydroxybutyryl-CoA. The invention also provides for a microbial cell engineered to express recombinant phaA gene and a recombinant phaB gene. In one embodiment, the microbial cell produces propylene. In another embodiment, the propylene is synthesized from acetyl-CoA. In still another embodiment, the terminal olefin synthesized in the microbial cell is selected from the group consisting of ethylene, propylene, butylenes, butadiene, isoprene, and 1-nonadecene. 
         [0010]    In one particular embodiment, the microbial cell recombinantly expresses a curM gene. In another particular embodiment, the microbial cell recombinantly expresses a nonA gene. 
         [0011]    In another embodiment of the present invention, an engineered microbial cell is provided, wherein the engineered microbial cell is selected from the group consisting of a bacterium, a yeast, and an algae, wherein the engineered microbial cell comprises one or more recombinant genes encoding a polypeptide comprising a sulfotransferase domain and/or a thioesterase domain, and wherein the engineered microbial cell synthesizes at least one terminal olefin. In a further embodiment, the bacterium is cyanobacterium. In another further embodiment, the bacterium is  E. Coli . In yet another embodiment, the bacterium is  Chlamydomonas reinhardtii . In still another embodiment, the bacterium is  Chlamydomonas reinhardtii . In one particular embodiment, the yeast is  S. cerevisiae.    
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0012]      FIG. 1 : Pathway for synthesis of propylene from 3-hydryxobutyryl-CoA or 3-hydroxybutyryl-ACP. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0013]    Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999). 
         [0014]    The following terms, unless otherwise indicated, shall be understood to have the following meanings: 
         [0015]    The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. 
         [0016]    The term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids. 
         [0017]    As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same microbial cell or progeny thereof) or exogenous (originating from a different microbial cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a microbial cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it. 
         [0018]    A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a microbial cell chromosome at a heterologous site and a nucleic acid construct present as an episome. 
         [0019]    The nucleic acids (also referred to as polynucleotides) of the present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids. 
         [0020]    The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)). 
         [0021]    The term “attenuate” as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional. In some instances a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is lessened such that the enzyme activity is not impacted by the presence of a compound. In other instances, an enzyme that has been altered to be less active can be referred to as attenuated. 
         [0022]    Deletion: 
         [0023]    The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together. 
         [0024]    Knock-Out: 
         [0025]    A gene whose level of functional expression or activity has been reduced to an undetectable levels. In some examples, a gene is knocked-out via deletion of some or all of its coding sequence. In other examples, a gene is knocked-out via introduction of one or more nucleotides into its open-reading frame, which results in translation of a non-sense or otherwise non-functional protein product. 
         [0026]    The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). 
         [0027]    “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest. 
         [0028]    The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. 
         [0029]    The term “recombinant microbial cell” (or simply “microbial cell” or “host cell”), as used herein, is intended to refer to a cell into which a recombinant nucleic acid molecule, such as, e.g., a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “microbial cell” or “host cell” as used herein. A recombinant microbial cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. 
         [0030]    The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function. 
         [0031]    The term “polypeptide” encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. 
         [0032]    The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment. 
         [0033]    The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal, an internal, and/or a carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long. 
         [0034]    A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as  125 I,  32 P,  35 S, and  3 H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference). 
         [0035]    The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein may comprise at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of any of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of an embodiment of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. 
         [0036]    The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W.H. Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present invention may be used to produce an equivalent effect and are therefore envisioned to be part of an embodiment of the present invention. 
         [0037]    The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. 
         [0038]    The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, a cytoplasmic domain, a thioesterase domain, and a sulfotransferase domain. 
         [0039]    The term thioesterase activity or “TE” refers to an enzymatic activity of a polypeptide which catalyzes the hydrolytic cleavage of energy-rich thioester bonds as in acetyl-CoA. This activity is useful in the catalytic conversion of 3-hydroxybutyryl-CoA or 3-hydroxybutyryl-ACP to propylene. 
         [0040]    The term sulfotransferase activity or “ST” refers to an enzymatic activity of a polypeptide which catalyzes the transfer of a sulfate group from one compound to the hydroxyl group of another. This activity is useful in the catalytic conversion of 3-hydroxybutyryl-CoA or 3-hydroxybutyryl-ACP to propylene. 
         [0041]    As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic. 
         [0042]    Biofuel: 
         [0043]    A biofuel is any fuel that derives from a biological source. Biofuel refers to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof. Preferably, liquid hydrocarbons are used. 
         [0044]    Hydrocarbon: 
         [0045]    The term generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, as well as plastics, waxes, solvents and oils. 
         [0046]    Terminal Olefin: 
         [0047]    a terminal olefin is an olefin (or alkene) having at least one carbon-carbon double bond located at the terminal end of the carbon chain backbone. Terminal olefins are unsaturated hydrocarbons. They can be straight chain, branched, and cyclic terminal olefins. 
         [0048]    Propylene or Propene: 
         [0049]    is an unsaturated organic compound having the chemical formula C3H6. It has one double bond, and is the second simplest member of the alkene class of hydrocarbons. 
         [0050]    Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. 
         [0051]    Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, in association with a numeric limitation, including a numeric range, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 
       Nucleic Acid Sequences 
       [0052]    Terminal olefins are chemical compounds that consist only of the elements carbon (C) and hydrogen (H) (i.e., hydrocarbons), containing at least carbon-carbon double bond (i.e., they are unsaturated compounds). Together, thioesterase (TE) and sulfotransferase (ST) enzymes function to synthesize terminal olefins, such as propylene from acetyl-CoA molecules and other precursors. 
         [0053]    Accordingly, an embodiment of the present invention provides isolated nucleic acid molecules for genes encoding TE and ST enzymes, and variants thereof. In one embodiment, the present invention provides an isolated nucleic acid molecule having a nucleic acid sequence comprising or consisting of a gene coding for TE and ST, and homologs, variants and derivatives thereof expressed in a host cell of interest. An embodiment of the present invention also provides a nucleic acid molecule comprising or consisting of a sequence which is a codon and expression optimized version of the TE and ST genes described herein. In a further embodiment, the present invention provides a nucleic acid molecule and homologs, variants and derivatives of the molecule comprising or consisting of a sequence which is a variant of the TE and ST gene having at least 76% sequence identity to a wild-type gene. The nucleic acid sequence can be preferably 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type gene. In one embodiment, the nucleic acid sequence encodes an enzyme selected from Tables 1-3 (SEQ ID NOS 4-104, respectively, in order of appearance). 
         [0054]    A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. 
         [0055]    Another embodiment of the invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. As defined above, and as is well known in the art, stringent hybridizations are performed at about 25° C. below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions, where the T m  is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Stringent washing is performed at temperatures about 5° C. lower than the T m  for the specific DNA hybrid under a particular set of conditions. 
         [0056]    Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides. 
         [0057]    As is well known in the art, enzyme activities can be measured in various ways. For example, the activity of the enzyme can be followed using chromatographic techniques, such as by high performance liquid chromatography. Chung and Sloan, J. Chromatogr. 371:71-81 (1986). As another alternative the activity can be indirectly measured by determining the levels of product made from the enzyme activity. These levels can be measured with techniques including aqueous chloroform/methanol extraction as known and described in the art (Cf M. Kates (1986)  Techniques of Lipidology; Isolation, analysis and identification of Lipids . Elsevier Science Publishers, New York (ISBN: 0444807322)). More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001).  Current practice of gas chromatography—mass spectrometry . New York, N.Y.: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix-Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G., R. O. Dunn, and M. O. Bagby. 1997. Biodiesel: The use of vegetable oils and their derivatives as alternative diesel fuels. Am. Chem. Soc. Symp. Series 666: 172-208), titration for determining free fatty acids (Komers, K., F. Skopal, and R. Stloukal. 1997. Determination of the neutralization number for biodiesel fuel production. Fett/Lipid 99(2): 52-54), enzymatic methods (Bailer, J., and K. de Hueber. 1991. Determination of saponifiable glycerol in “bio-diesel.” Fresenius J. Anal. Chem. 340(3): 186), physical property-based methods, wet chemical methods, etc. can be used to analyze the levels and the identity of the product produced by the organisms of an embodiment of the present invention. Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art. 
       Plasmids 
       [0058]    Plasmids relevant to genetic engineering typically include at least two functional elements 1) an origin of replication enabling propagation of the DNA sequence in the host organism, and 2) a selective marker (for example an antibiotic resistance marker conferring resistance to ampicillin, kanamycin, zeocin, chloramphenicol, tetracycline, spectinomycin, and the like). Plasmids are often referred to as “cloning vectors” when their primary purpose is to enable propagation of a desired heterologous DNA insert. Plasmids can also include cis-acting regulatory sequences to direct transcription and translation of heterologous DNA inserts (for example, promoters, transcription terminators, ribosome binding sites); such plasmids are frequently referred to as “expression vectors.” When plasmids contain functional elements that allow for propagation in more than one species, such plasmids are referred to as “shuttle vectors.” Shuttle vectors are well known to those in the art. For example, pSE4 is a shuttle vector that allows propagation in  E. coli  and  Synechococcus  [Maeda S, Kawaguchi Y, Ohy T, and Omata T. J. Bacteriol. (1998). 180:4080-4088]. Shuttle vectors are particularly useful in one embodiment of the present invention to allow for facile manipulation of genes and regulatory sequences. 
       Vectors 
       [0059]    Also provided are vectors, including expression vectors and cloning vectors, which comprise the above nucleic acid molecules of an embodiment of the present invention. In a first embodiment, the vectors include the isolated nucleic acid molecules described above. In an alternative embodiment, the vectors include the above-described nucleic acid molecules operably linked to one or more expression control sequences. The vectors of the instant invention may thus be used to express an ST and/or TE polypeptide contributing to polypropylene producing activity by a host cell. 
         [0060]    Exemplary vectors of the invention include any of the vectors expressing a thioesterase or sulfotranserase. A gene expressing a thioesterase or sulfotransferase are assembled and inserted into a suitable vector, e.g. pJB5, as described in WO2009/111513, herein incorporated in its entirety by reference. The invention also provides other vectors such as pJB161, as described in WO2009/062190 and U.S. Pat. No. 7,785,861, herein incorporated in their entirety by reference, which are capable of receiving nucleic acid sequences of the invention. Vectors such as pJB161 comprise sequences which are homologous with sequences that are present in plasmids which are endogenous to certain photosynthetic microorganisms (e.g., plasmids pAQ7 or pAQ1 of certain  Synechococcus  species). Recombination between pJB161 and the endogenous plasmids in vivo yield engineered microbes expressing the genes of interest from their endogenous plasmids. Alternatively, vectors can be engineered to recombine with the host cell chromosome, or the vector can be engineered to replicate and express genes of interest independent of the host cell chromosome or any of the host cell&#39;s endogenous plasmids. 
         [0061]    Vectors useful for expression of nucleic acids in prokaryotes are well known in the art. 
       Isolated Polypeptides 
       [0062]    According to another aspect of the present invention, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules are provided. In one embodiment, isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids. 
         [0063]    The polypeptides of an embodiment of the present invention also include fusions between the above-described polypeptide sequences and heterologous polypeptides. The heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins. Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region. 
       Host Cell Transformants 
       [0064]    In another aspect of the present invention, host cells transformed with the nucleic acid molecules or vectors of an embodiment of the present invention, and descendants thereof, are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences of an embodiment of the present invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the present invention, the nucleic acids have been integrated into the genome of the host cells. 
         [0065]    In a preferred embodiment, the host cell comprises one or more ST and/or TE encoding nucleic acids which express ST and/or TE activity in the host cell. 
         [0066]    In an alternative embodiment, the host cells of an embodiment of the present invention are mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present invention so that the activity of the ST and/or TE protein(s) in the host cell is reduced or eliminated compared to a host cell lacking the mutation. 
       Selected or Engineered Microorganisms for the Production of Carbon-Based Products of Interest 
       [0067]    Microorganism: Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” and “microbes” are used interchangeably with the term microorganism. 
         [0068]    A variety of host organisms can be transformed to produce a product of interest. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria. 
         [0069]    Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include hyperthermophiles, which grow at or above 80° C. such as  Pyrolobus fumarii ; thermophiles, which grow between 60-80° C. such as  Synechococcus lividis ; mesophiles, which grow between 15-60° C. and psychrophiles, which grow at or below 15° C. such as  Psychrobacter  and some insects. Radiation tolerant organisms include  Deinococcus radiodurans . Pressure tolerant organisms include piezophiles, which tolerate pressure of 130 MPa. Weight tolerant organisms include barophiles. Hypergravity (e.g., &gt;1 g) hypogravity (e.g., &lt;1 g) tolerant organisms are also contemplated. Vacuum tolerant organisms include tardigrades, insects, microbes and seeds. Dessicant tolerant and anhydrobiotic organisms include xerophiles such as  Artemia salina ; nematodes, microbes, fungi and lichens. Salt tolerant organisms include halophiles (e.g., 2-5 M NaCl)  Halobacteriacea  and  Dunaliella salina . pH tolerant organisms include alkaliphiles such as  Natronobacterium, Bacillus firmus  OF4,  Spirulina  spp. (e.g., pH&gt;9) and acidophiles such as  Cyanidium caldarium, Ferroplasma  sp. (e.g., low pH). Anaerobes, which cannot tolerate O 2  such as  Methanococcus jannaschii ; microaerophils, which tolerate some O 2  such as  Clostridium  and aerobes, which require O 2  are also contemplated. Gas tolerant organisms, which tolerate pure CO 2  include  Cyanidium caldarium  and metal tolerant organisms include metalotolerants such as  Ferroplasma acidarmanus  (e.g., Cu, As, Cd, Zn),  Ralstonia  sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael.  Life on the Edge: Amazing Creatures Thriving in Extreme Environments . New YorK: Plenum (1998) and Seckbach, J. “Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds.,  Astronomical and Biochemical Origins and the Search for Life in the Universe , p. 511. Milan: Editrice Compositori (1997). 
         [0070]    Plants include but are not limited to the following genera:  Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia  and  Zea.    
         [0071]    Algae and cyanobacteria include but are not limited to the following genera: 
         [0072]      Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis , and  Zygonium.    
         [0073]    Green non-sulfur bacteria include but are not limited to the following genera:  Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus , and  Thermomicrobium.    
         [0074]    Green sulfur bacteria include but are not limited to the following genera: 
         [0075]      Chlorobium, Clathrochloris , and  Prosthecochloris.    
         [0076]    Purple sulfur bacteria include but are not limited to the following genera:  Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus , and  Thiocystis,    
         [0077]    Purple non-sulfur bacteria include but are not limited to the following genera:  Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio , and  Roseospira.    
         [0078]    Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as  Nitrobacteraceae  sp.,  Nitrobacter  sp.,  Nitrospina  sp.,  Nitrococcus  sp.,  Nitrospira  sp.,  Nitrosomonas  sp.,  Nitrosococcus  sp.,  Nitrosospira  sp.,  Nitrosolobus  sp.,  Nitrosovibrio  sp.; colorless sulfur bacteria such as,  Thiovulum  sp.,  Thiobacillus  sp.,  Thiomicrospira  sp.,  Thiosphaera  sp.,  Thermothrix  sp.; obligately chemolithotrophic hydrogen bacteria such as  Hydrogenobacter  sp., iron and manganese-oxidizing and/or depositing bacteria such as  Siderococcus  sp., and magnetotactic bacteria such as  Aquaspirillum  sp. 
         [0079]    Archaeobacteria include but are not limited to methanogenic archaeobacteria such as  Methanobacterium  sp.,  Methanobrevibacter  sp.,  Methanothermus  sp.,  Methanococcus  sp.,  Methanomicrobium  sp.,  Methanospirillum  sp.,  Methanogenium  sp.,  Methanosarcina  sp.,  Methanolobus  sp.,  Methanothrix  sp.,  Methanococcoides  sp.,  Methanoplanus  sp.; extremely thermophilic S°-Metabolizers such as  Thermoproteus  sp.,  Pyrodictium  sp.,  Sulfolobus  sp.,  Acidianus  sp. and other microorganisms such as,  Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces  sp.,  Ralstonia  sp.,  Rhodococcus  sp.,  Corynebacteria  sp.,  Brevibacteria  sp.,  Mycobacteria  sp., and oleaginous yeast. 
         [0080]    HyperPhotosynthetic conversion requires extensive genetic modification; thus, in preferred embodiments the parental photoautotrophic organism can be transformed with exogenous DNA. 
         [0081]    Preferred organisms for HyperPhotosynthetic conversion include:  Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus , and  Zea mays  (plants),  Botryococcus braunii, Chlamydomonas reinhardtii  and  Dunaliela salina  (algae),  Synechococcus  sp PCC 7002,  Synechococcus  sp. PCC 7942,  Synechocystis  sp. PCC 6803, and  Thermosynechococcus elongatus  BP-1 (cyanobacteria),  Chlorobium tepidum  (green sulfur bacteria),  Chloroflexus auranticus  (green non-sulfur bacteria),  Chromatium tepidum  and  Chromatium vinosum  (purple sulfur bacteria),  Rhodospirillum rubrum, Rhodobacter capsulatus , and  Rhodopseudomonas palusris  (purple non-sulfur bacteria). 
         [0082]    Yet other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862. 
         [0083]    Still, other suitable organisms include microorganisms that can be engineered to fix carbon dioxide bacteria such as  Escherichia coli, Acetobacter aceti, Bacillus subtilis , yeast and fungi such as  Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens , or  Zymomonas mobilis.    
         [0084]    A common theme in selecting or engineering a suitable organism is autotrophic fixation of CO 2  to products. This would cover photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of CO 2  fixation; Calvin cycle, acetyl CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups ofprokaryotes. The CO 2  fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. Fuchs, G. 1989. Alternative pathways of autotrophic CO 2  fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer-Verlag, Berlin, Germany. The reductive pentose phosphate cycle (Calvin-Bassham-Benson cycle) represents the CO 2  fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria. 
         [0085]    The host cell of one embodiment of the present invention is preferably  Escherichia coli, Synechococcus, Thermosynechococcus, Synechocystis, Klebsiella oxytoca , or  Saccharomyces cerevisiae  but other prokaryotic, archaea and eukaryotic host cells including those of the cyanobacteria are also encompassed within the scope of the present invention. 
       Hydroxyacyl Substrates 
       [0086]    The compositions and methods described herein can be used to produce olefins (e.g., terminal olefins) from hydroxyacyl substrates. While not wishing to be bound by theory it is believed that the polypeptides described herein produce olefins from hydroxyacyl substrates via a sulfotransferase and thioesterase mechanism. Thus, olefins having particular branching patterns, levels of saturation, and carbon chain length can be produced from hydroxyacyl substrates having those particular characteristics. Accordingly, each step within a hydroxyacyl related pathway can be modified to produce or overproduce a hydroxyacyl substrate of interest. 
       Producing Terminal Olefins Using Cell-Free Methods 
       [0087]    Some methods described herein, a terminal olefin can be produced using a purified polypeptide described herein and a hydroxyacyl substrate. For example, a host cell can be engineered to express a polypeptide (e.g. a NonA polypeptide or a variant thereof) as described herein. The host cell can be cultured under conditions suitable to allow expression of the polypeptide. Cell free extracts can then be generated using known methods. For example, the host cells can be lysed using detergents or by sonication. The expressed polypeptides can be purified using known methods. After obtaining the cell free extracts, hydroxyacyl substrates described herein can be added to the cell free extracts and maintained under conditions to allow conversion of hydroxyacyl substrates to terminal olefins. The terminal olefins can be separated and purified using known techniques. 
         [0088]    The following examples are for illustrative purposes and are not intended to limit the scope of the present invention. 
       Example 1 
     A Pathway for the Enzymatic Synthesis of Terminal Olefins from 3-Hydroxyacyl Substrates 
       [0089]    The nonA gene in  Synechococcus elongatus  PCC 7002 has been discovered by us to be responsible for synthesis of 1-nonadecene and other long-chain terminal olefins, as described in PCT/US2010/039558, herein incorporated by reference in its entirety. This newly discovered enzymatic activity is attributed to ST and TE domains present in the enzyme expressed by this gene. In this example, we express ST and TE domains of a protein such as  L. majuscula  CurM or  S. elongatus  PCC 7002 NonA in a host cell to convert 3-hydroxyacyl substrates to the corresponding terminal olefins, e.g. propylene. 
       Example 2 
     A Pathway for the Enzymatic Synthesis of Propylene 
       [0090]    In this example, we use recombinant or endogenous ST and TE activity to convert 3-hydroxybutyryl-ACP or 3-hydroxybutyryl-CoA to propylene and CO 2  with the help of the cofactor 3′-phosphate 5′-phosphosulfate (PAPS), which occurs widely in bacterial and other biological systems ( FIG. 1 ). 
         [0091]    To obtain 3-hydroxybutyryl-CoA, we express  R. eutropha  phaA and phaB in the host cell, whose gene products together convert 2 acetyl-CoA molecules to 3-hydroxybutyryl-CoA and CoA, using NADPH as a cofactor. 
         [0092]    To obtain 3-hydroxybutyryl-ACP, we utilize a host with attenuated 3-hydroxyacyl-ACP dehydratase (EC 4.2.1.59 and/or EC 4.2.1.58) activity while feeding long-chain fatty acids to enable lipid synthesis. In an alternative embodiment, the 3-hydroxyacyl-ACP dehydratase is placed under inducible control and expressed only under growth conditions. This allows fatty acid biosynthesis to proceed only to 3-hydroxybutyryl-ACP while still allowing the cell to grow. In this way, one obtains a pathway from acetyl-CoA to propylene. 
       Example 3 
     Homologous ST and TE Domains 
       [0093]    The sequences of the ST and TE domains of the  Synechococcus elongatus  sp. PCC7002 NonA protein (SEQ ID NO:1) were used to perform an amino acid sequence search for homologous proteins using BLAST. Proteins homologous to the region of the protein comprising both ST and TE domains are listed in Table 1 (SEQ ID NOS 4-11, respectively, in order of appearance). Sequences homologous to only the NonA ST domain protein sequence (SEQ ID NO:2) are listed in Table 2 (SEQ ID NOS 12-19, respectively, in order of appearance). Sequences homologous to only the NonA TE domain protein sequence (SEQ ID NO:3) are listed in Table 3 (SEQ ID NOS 20-104, respectively, in order of appearance). At least one of the protein sequences of Tables 1-3 (SEQ ID NOS 4-104, respectively, in order of appearance) is engineered into a host cell, e.g. cyanobacterium, according to standard genetic engineering techniques. The engineered host cell has an increased capacity to synthesize terminal olefins, e.g. propylene. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Proteins showing homology to both ST and TE domains of NonA. 
               
             
          
           
               
                 SEQ ID NO: 
                 Protein ID 
                 GenBank-annotated function 
                 Organism 
               
               
                   
               
             
          
           
               
                 4 
                 YP_001734428.1 
                 polyketide synthase 
                   Synechococcus  sp. PCC 7002 
               
               
                 5 
                 YP_002377174.1 
                 beta-ketoacyl synthase 
                   Cyanothece  sp. PCC 7424 
               
               
                 6 
                 YP_003887107.1 
                 beta-ketoacyl synthase 
                   Cyanothece  sp. PCC 7822 
               
               
                 7 
                 ACV42478.1 
                 polyketide synthase 
                   Lyngbya   majuscula  19L 
               
               
                 8 
                 AAT70108.1 
                 CurM 
                 
                   Lyngbya 
                   majuscula 
                 
               
               
                 9 
                 YP_610919.1 
                 polyketide synthase 
                   Pseudomonas   entomophila  L48 
               
               
                 10 
                 YP_003265308.1 
                 KR domain protein 
                   Haliongium   ochraceum  DSM 
               
               
                   
                   
                   
                 14365 
               
               
                 11 
                 XP_002507643.1 
                 modular polyketide synthase 
                   Micromonas  sp. RCC299 
               
               
                   
                   
                 type I 
                   
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Proteins showing homology to only the ST domain of NonA. 
               
             
          
           
               
                 SEQ ID NO: 
                 Protein ID 
                 GenBank-annotated function 
                 Organism 
               
               
                   
               
             
          
           
               
                 12 
                 YP_001062692.1 
                 CurM 
                   Burkholderia   pseudomallei  668 
               
               
                 13 
                 ABW84363.1 
                 OciA 
                   Planktothrix   agardhii  NIES-205 
               
               
                 14 
                 ABI26077.1 
                 OciA 
                   Planktothrix   agardhii  NIVA-CYA 
               
               
                   
                   
                   
                 116 
               
               
                 15 
                 YP_003137597.1 
                 amino acid adenylation 
                   Cyanothece  sp. PCC 8802 
               
               
                   
                   
                 domain protein 
                   
               
               
                 16 
                 YP_002372038.1 
                 amino acid adenylation 
                   Cyanothece  sp. PCC 8801 
               
               
                   
                   
                 domain protein 
                   
               
               
                 17 
                 XP_003074830.1 
                 COG3321: Polyketide 
                 
                   Ostreococcus 
                   tauri 
                 
               
               
                   
                   
                 synthase modules and related 
                   
               
               
                   
                   
                 proteins (ISS) 
                   
               
               
                 18 
                 XP_001416378.1 
                 polyketide synthase 
                 
                   Ostreococcus 
                   lucimarinus 
                 
               
               
                   
                   
                   
                 CCE9901 
               
               
                 19 
                 ZP_03631565.1 
                 amino acid adenylation 
                 bacterium Ellin514 
               
               
                   
                   
                 domain protein 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Proteins showing homology to only the TE domain of NonA. 
               
             
          
           
               
                 SEQ ID NO: 
                 Protein ID 
                 GenBank-annotated function 
                 Organism 
               
               
                   
               
             
          
           
               
                 20 
                 YP_001734428.1 
                 polyketide synthase 
                   Synechococcus  sp. PCC 
               
               
                   
                   
                   
                 7002 
               
               
                 21 
                 AAC14106.1 
                 epoxide hydroxylase 
                   Synechococcus  sp. PCC 
               
               
                   
                   
                   
                 7002 
               
               
                 22 
                 YP_433651.1 
                 alpha/beta superfamily 
                   Hahella   chejuensis  KCTC 
               
               
                   
                   
                 hydrolase/acyltransferase 
                 2396 
               
               
                 23 
                 YP_001769292.1 
                 alpha/beta hydrolase fold 
                   Methylobacterium  sp. 4- 
               
               
                   
                   
                   
                 46 
               
               
                 24 
                 YP_003269090.1 
                 alpha/beta hydrolase fold protein 
                 
                   Haliongium 
                   ochraceum 
                 
               
               
                   
                   
                   
                 DSM 14365 
               
               
                 25 
                 ZP_01916760.1 
                 Alpha/beta hydrolase fold protein 
                   Limnobacter  sp. MED105 
               
               
                 26 
                 YP_933620.1 
                 hydrolase or acytransferase 
                   Azoarcus  sp. BH72 
               
               
                 27 
                 YP_158988.1 
                 putative hydrolase 
                 
                   Aromatoleum 
                   aromaticum 
                 
               
               
                   
                   
                   
                 EbN1 
               
               
                 28 
                 YP_003776671.1 
                 hydrolase 
                 
                   Herbaspirillum 
                 
               
               
                   
                   
                   
                   seropedicae  SmR1 
               
               
                 29 
                 BAI49930.1 
                 putative esterase 
                 uncultured microorganism 
               
               
                 30 
                 YP_662370.1 
                 alpha/beta hydrolase fold 
                 
                   Pseudoalteromonas 
                 
               
               
                   
                   
                   
                   atlantica  T6c 
               
               
                 31 
                 ZP_01459983.1 
                 lipase A 
                 
                   Stigmatella 
                   aurantiaca 
                 
               
               
                   
                   
                   
                 DW4/3-1 
               
               
                 32 
                 YP_634109.1 
                 alpha/beta fold family hydrolase  
                   Myxococcus   xanthus  DK 
               
               
                   
                   
                   
                 1622 
               
               
                 33 
                 ZP_01615147.1 
                 alpha/beta hydrolase 
                 marine gamma 
               
               
                   
                   
                   
                 proteobacterium 
               
               
                   
                   
                   
                 HTCC2143 
               
               
                 34 
                 YP_001352966.1 
                 alpha/beta fold family hydrolase 
                   Janthinobacterium  sp. 
               
               
                   
                   
                   
                 Marseille 
               
               
                 35 
                 ZP_01307598.1 
                 hydrolase, alpha/beta fold family 
                   Oceanobacter  sp. RED65 
               
               
                   
                   
                 protein 
                   
               
               
                 36 
                 YP_001100441.1 
                 putative hydrolase protein 
                 
                   Herminiimonas 
                 
               
               
                   
                   
                   
                 
                   arsenicoxydans 
                 
               
               
                 37 
                 EFP65715.1 
                 alpha/beta hydrolase family protein 
                   Ralstonia  sp. 5_7_47FAA 
               
               
                 38 
                 YP_002981038.1 
                 alpha/beta hydrolase fold protein 
                   Ralstonia   pickettii  12D 
               
               
                 39 
                 YP_001898558.1 
                 alpha/beta hydrolase fold 
                   Ralstonia   pickettii  12J 
               
               
                 40 
                 YP_001172415.1 
                 hydrolase 
                 
                   Pseudomonas 
                   stutzeri 
                 
               
               
                   
                   
                   
                 A1501 
               
               
                 41 
                 YP_002354112.1 
                 alpha/beta hydrolase fold protein 
                   Thauera  sp. MZ1T 
               
               
                 42 
                 ZP_05040720.1 
                 hydrolase, alpha/beta fold family,  
                   Alcanivorax  sp. DG881 
               
               
                   
                   
                 putative 
                   
               
               
                 43 
                 YP_001021961.1 
                 putative hydrolase protein 
                 
                   Methylibium 
                 
               
               
                   
                   
                   
                   petroleiphilum  PM1 
               
               
                 44 
                 YP_002030374.1 
                 alpha/beta hydrolase fold 
                 
                   Stenotrophomonas 
                 
               
               
                   
                   
                   
                   maltophilia  R551-3 
               
               
                 45 
                 ZP_01126880.1 
                 Alpha/beta hydrolase fold protein 
                   Nitrococcus   mobilis  Nb- 
               
               
                   
                   
                   
                 231 
               
               
                 46 
                 YP_001974273.1 
                 putative alpha/beta fold hydrolase 
                 
                   Stenotrophomonas 
                 
               
               
                   
                   
                 family protein 
                   maltophilia  K279a 
               
               
                 47 
                 YP_286430.1 
                 Alpha/beta hydrolase fold 
                 
                   Dechloromonas 
                   aromatica 
                 
               
               
                   
                   
                   
                 RCB 
               
               
                 48 
                 YP_001990203.1 
                 alpha/beta hydrolase fold 
                 
                   Rhodopseudomonas 
                 
               
               
                   
                   
                   
                   palustris  TIE-1 
               
               
                 49 
                 YP_917027.1 
                 alpha/beta hydrolase fold 
                 
                   Paracoccus 
                   denitrificans 
                 
               
               
                   
                   
                   
                 PD1222 
               
               
                 50 
                 YP_002005206.1 
                 putative Alpha/beta fold hydrolase  
                 
                   Cupriavidus 
                   taiwanensis 
                 
               
               
                 51 
                 YP_283592.1 
                 Alpha/beta hydrolase fold 
                 
                   Dechloromonas 
                   aromatica 
                 
               
               
                   
                   
                   
                 RCB 
               
               
                 52 
                 YP_001349005.1 
                 putative hydrolase 
                 
                   Pseudomonas 
                   aeruginosa 
                 
               
               
                   
                   
                   
                 PA7 
               
               
                 53 
                 YP_001187947.1 
                 alpha/beta hydrolase fold 
                 
                   Pseudomonas 
                   mendocina 
                 
               
               
                   
                   
                   
                 ymp 
               
               
                 54 
                 ZP_04576152.1 
                 hydrolase 
                 
                   Oxalobacter 
                   formigenes 
                 
               
               
                   
                   
                   
                 HOxBLS 
               
               
                 55 
                 NP_250313.1 
                 probable hydrolase 
                 
                   Pseudomonas 
                   aeruginosa 
                 
               
               
                   
                   
                   
                 PAO1 
               
               
                 56 
                 NP_900963.1 
                 hydrolase 
                 
                   Chromobacterium 
                 
               
               
                   
                   
                   
                   violaceum  ATCC 12472 
               
               
                 57 
                 AAT50924.1 
                 PA1622 
                 synthetic construct 
               
               
                 58 
                 YP_725707.1 
                 alpha/beta superfamily 
                   Ralstonia   eutropha  H16 
               
               
                   
                   
                 hydrolase/acyltransferase 
                   
               
               
                 59 
                 YP_001554328.1 
                 alpha/beta hydrolase fold 
                   Shewanella   baltica  OS195 
               
               
                 60 
                 YP_002441288.1 
                 putative hydrolase 
                 
                   Pseudomonas 
                   aeruginosa 
                 
               
               
                   
                   
                   
                 LESB58 
               
               
                 61 
                 YP_693203.1 
                 hydrolase 
                 
                   Alcanivorax 
                   borkumensis 
                 
               
               
                   
                   
                   
                 SK2 
               
               
                 62 
                 YP_002798221.1 
                 alpha/beta hydrolase 
                   Azotobacter   vinelandii  DJ 
               
               
                 63 
                 NP_001079604.1 
                 serine hydrolase-like 2 
                 
                   Xenopus 
                   laevis 
                 
               
               
                 64 
                 NP_946347.1 
                 Alpha/beta hydrolase fold 
                 
                   Rhodopseudomonas 
                 
               
               
                   
                   
                   
                   palustris  CGA009 
               
               
                 65 
                 YP_870022.1 
                 alpha/beta hydrolase fold 
                   Shewanella  sp. ANA-3 
               
               
                 66 
                 YP_295320.1 
                 Alpha/beta hydrolase fold 
                 
                   Ralstonia 
                   eutropha 
                 
               
               
                   
                   
                   
                 JMP134 
               
               
                 67 
                 YP_001982425.1 
                 hydrolase, alpha/beta fold family 
                 
                   Cellvibrio 
                   japonicus 
                 
               
               
                   
                   
                   
                 Ueda107 
               
               
                 68 
                 YP_963643.1 
                 alpha/beta hydrolase fold 
                   Shewanella  sp. W3-18-1 
               
               
                 69 
                 ZP_06358651.1 
                 alpha/beta hydrolase fold protein 
                 
                   Rhodopseudomonas 
                 
               
               
                   
                   
                   
                   palustris  DX-1 
               
               
                 70 
                 YP_001366096.1 
                 alpha/beta hydrolase fold 
                   Shewanella   baltica  OS185 
               
               
                 71 
                 ZP_01707636.1 
                 alpha/beta hydrolase fold 
                 
                   Shewanella 
                   putrefaciens 
                 
               
               
                   
                   
                   
                 200 
               
               
                 72 
                 YP_734308.1 
                 alpha/beta hydrolase fold 
                   Shewanella  sp. MR-4 
               
               
                 73 
                 ZP_04957287.1 
                 hydrolase 
                 gamma proteobacterium 
               
               
                   
                   
                   
                 NOR51-B 
               
               
                 74 
                 NP_718168.1 
                 alpha/beta fold family hydrolase 
                 
                   Shewanella 
                   oneidensis 
                 
               
               
                   
                   
                   
                 MR-1 
               
               
                 75 
                 YP_003146580.1 
                 alpha/beta hydrolase fold protein 
                   Kangiella   koreensis  DSM 
               
               
                   
                   
                   
                 16069 
               
               
                 76 
                 YP_568320.1 
                 alpha/beta hydrolase fold 
                 
                   Rhodopseudomonas 
                 
               
               
                   
                   
                   
                   palustris  BisB5 
               
               
                 77 
                 YP_001183284.1 
                 alpha/beta hydrolase fold 
                 
                   Shewanella 
                   putrefaciens 
                 
               
               
                   
                   
                   
                 CN-32 
               
               
                 78 
                 ZP_05134273.1 
                 hydrolase of the alpha/beta fold  
                   Stenotrophomonas  sp. 
               
               
                   
                   
                 superfamily 
                 SKA14 
               
               
                 79 
                 YP_003545632.1 
                 putative alpha/beta hydrolase 
                 
                   Sphingobium 
                   japonicum 
                 
               
               
                   
                   
                   
                 UT26S 
               
               
                 80 
                 YP_002358347.1 
                 alpha/beta hydrolase fold protein 
                   Shewanella   baltica  OS223 
               
               
                 81 
                 YP_856727.1 
                 alpha/beta fold family hydrolase  
                 
                   Aeromonas 
                   hydrophila 
                 
               
               
                   
                   
                   
                 subsp.  hydrophila  ATCC 
               
               
                   
                   
                   
                 7966 
               
               
                 82 
                 XP_003055946.1 
                 predicted protein 
                 
                   Micromonas 
                   pusilla 
                 
               
               
                   
                   
                   
                 CCMP1545 
               
               
                 83 
                 ZP_01616002.1 
                 putative hydrolase 
                 marine gamma 
               
               
                   
                   
                   
                 proteobacterium 
               
               
                   
                   
                   
                 HTCC2143 
               
               
                 84 
                 YP_001411669.1 
                 alpha/beta hydrolase fold 
                 
                   Parvibaculum 
                 
               
               
                   
                   
                   
                   lavamentivorans  DS-1 
               
               
                 85 
                 ZP_07392985.1 
                 alpha/beta hydrolase fold protein 
                   Shewanella   baltica  OS183 
               
               
                 86 
                 YP_001050238.1 
                 alpha/beta hydrolase fold 
                   Shewanella   baltica  OS155 
               
               
                 87 
                 YP_002553684.1 
                 alpha/beta hydrolase fold protein 
                   Acidovorax   ebreus  TPSY 
               
               
                 88 
                 YP_003165824.1 
                 alpha/beta hydrolase fold protein 
                 
                   Candidatus 
                 
               
               
                   
                   
                   
                 
                   Accumulibacter 
                 
               
               
                   
                   
                   
                   phosphatis  clade IIA str. 
               
               
                   
                   
                   
                 UW-1 
               
               
                 89 
                 YP_001141910.1 
                 alpha/beta fold family hydrolase 
                 
                   Aeromonas 
                   salmonicida 
                 
               
               
                   
                   
                   
                 subsp.  salmonicida  A449 
               
               
                 90 
                 ZP_04579173.1 
                 hydrolase 
                 
                   Oxalobacter 
                   formigenes 
                 
               
               
                   
                   
                   
                 OXCC13 
               
               
                 91 
                 YP_001502304.1 
                 alpha/beta hydrolase fold 
                 
                   Shewanella 
                   pealeana 
                 
               
               
                   
                   
                   
                 ATCC 700345 
               
               
                 92 
                 YP_484670.1 
                 Alpha/beta hydrolase 
                 
                   Rhodopseudomonas 
                 
               
               
                   
                   
                   
                   palustris  HaA2 
               
               
                 93 
                 YP_001615653.1 
                 putative hydrolase 
                   Sorangium   cellulosum  ‘So 
               
               
                   
                   
                   
                 ce 56’ 
               
               
                 94 
                 YP_003752880.1 
                 putative Alpha/beta fold hydrolase 
                 
                   Ralstonia 
                   solanacearum 
                 
               
               
                   
                   
                   
                 PS107 
               
               
                 95 
                 XP_002192434.1 
                 PREDICTED: serine hydrolase-like 2 
                 
                   Taeniopygia 
                   guttata 
                 
               
               
                 96 
                 YP_235108.1 
                 Alpha/beta hydrolase fold 
                   Pseudomonas   syringae  pv. 
               
               
                   
                   
                   
                 syringae B728a 
               
               
                 97 
                 YP_002795270.1 
                 Probable hydrolase 
                 
                   Laribacter 
                   hongkongensis 
                 
               
               
                   
                   
                   
                 HLHK9 
               
               
                 98 
                 XP_001749708.1 
                 hypothetical protein 
                   Monosiga   brevicollis  MX1 
               
               
                 99 
                 YP_274221.1 
                 lipase, putative 
                   Pseudomonas   syringae  pv. 
               
               
                   
                   
                   
                 phaseolicola 1448A 
               
               
                 100 
                 ZP_02374233.1 
                 hydrolase, alpha/beta fold family 
                 
                   Burkholderia 
                   thailandensis 
                 
               
               
                   
                   
                 protein 
                 TXDOH 
               
               
                 101 
                 YP_003073941.1 
                 alpha/beta hydrolase family protein 
                 
                   Teredinibacter 
                   turnerae 
                 
               
               
                   
                   
                   
                 T7901 
               
               
                 102 
                 ZP_00945280.1 
                 Esterase 
                 
                   Ralstonia 
                   solanacearum 
                 
               
               
                   
                   
                   
                 UW551 
               
               
                 103 
                 YP_002253305.1 
                 hydrolase or acyltransferase 
                 
                   Ralstonia 
                   solanacearum 
                 
               
               
                   
                   
                 (alpha/beta hydrolase superfamily) 
                 MoIK2 
               
               
                   
                   
                 protein 
                   
               
               
                 104 
                 YP_003746098.1 
                 putative Alpha/beta fold hydrolase 
                 
                   Ralstonia 
                   solanacearum 
                 
               
               
                   
                   
                   
                 CFBP2957 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
             
           
               
                 INFORMAL SEQUENCE LISTING 
                   
               
               
                 SEQ ID NO: 1 
                   
               
               
                   Synechococcus elongatus  NonA (SYNPCC7002_A1173) 
                   
               
               
                 Protein sequence 
               
               
                 ST domain is  underlined , TE domain is  in bold . 
               
               
                 MASWSHPQFEKEVHHHHHHGAVGQFANFVDLLQYRAKLQARKTVFSFLADGEAESAALTYGELDQKAQAI 
               
               
                   
               
               
                 AAFLQANQAQGQRALLLYPPGLEFIGAFLGCLYAGVVAVPAYPPRPNKSFDRLHSIIQDAQAKFALTTTE 
               
               
                   
               
               
                 LKDKIADRLEALEGTDFHCLATDQVELISGKNWQKPNISGTDLAFLQYTSGSTGDPKGVMVSHHNLIHNS 
               
               
                   
               
               
                 GLINQGFQDTEASMGVSWLPPYHDMGLIGGILQPIYVGATQILMPPVAFLQRPFRWLKAINDYRVSTSGA 
               
               
                   
               
               
                 PNFAYDLCASQITPEQIRELDLSCWRLAFSGAEPIRAVTLENFAKTFATAGFQKSAFYPCYGMAETTLIV 
               
               
                   
               
               
                 SGGNGRAQLPQEIIVSKQGIEANQVRPAQGTETTVTLVGSGEVIGDQIVKIVDPQALTECTVGEIGEVWV 
               
               
                   
               
               
                 KGESVAQGYWQKPDLTQQQFQGNVGAETGFLRTGDLGFLQGGELYITGRLKDLLIIRGRNHYPQDIELTV 
               
               
                   
               
               
                 EVAHPALRQGAGAAVSVDVNGEEQLVIVQEVERKYARKLNVAAVAQAIRGAIAAEHQLQPQAICFIKPGS 
               
               
                   
               
               
                 IPKTSSGKIRRHACKAGFLDGSLAVVGEWQPSHQKEGKGIGTQAVTPSTTTSTNFPLPDQHQQQIEAWLK 
               
               
                   
               
               
                 DNIAHRLGITPQQLDETEPFASYGLDSVQAVQVTADLEDWLGRKLDPTLAYDYPTIRTLAQFLVQGNQAL 
               
               
                   
               
               
                 EKIPQVPKIQGKEIAVVGLSCRFPQADNPEAFWELLRNGKDGVRPLKTRWATGEWGGFLEDIDQFEPQFF 
               
               
                   
               
               
                 GISPREAEQMDPQQRLLLEVTWEALERANIPAESLRHSQTGVFVGISNSDYAQLQVRENNPINPYMGTGN 
               
               
                   
               
               
                 AHSIAANRLSYFLDLRGVSLSIDTACSSSLVAVHLACQSLINGESELAIAAGVNLILTPDVTQTFTQAGM 
               
               
                   
               
               
                 MSKTGRCQTFDAEADGYVRGEGCGVVLLKPLAQAERDGDNILAVIHGSAVNQDGRSNGLTAPNGRSQQAV 
               
               
                   
               
               
                 IRQALAQAGITAADLAYLEAHGTGTPLGDPIEINSLKAVLQTAQREQPCVVGSVKTNIGHLEAAAGIAGL 
               
               
                   
               
               
                 IKVILSLEHGMIPQHLHFKQLNPRIDLDGLVTIASKDQPWSGGSQKRFAGVSSFGFGGTNAHVIVGDYAQ 
               
               
                   
               
               
                 QKSPLAPPATQDRPWHLLTLSAKNAQALNALQKSYGDYLAQHPSVDPRDLCLSANTGRSPLKERRFFVFK 
               
               
                   
               
               
                 QVADLQQTLNQDFLAQPRLSSPAKIAFLFTGQGSQYYGMGQQLYQTSPVFRQVLDECDRLWQTYSPEAPA 
               
               
                   
               
               
                 LTDLLYGNHNPDLVHETVYTQPLLFAVEYAIAQLWLSWGVTPDFCMGHSVGEYVAACLAGVFSLADGMKL 
               
               
                   
               
               
                 ITARGKLMHALPSNGSMAAVFADKTVIKPYLSEHLTVGAENGSHLVLSGKTPCLEASIHKLQSQGIKTKP 
               
               
                   
               
               
                 LKVSHAFHSPLMAPMLAEFREIAEQITFHPPRIPLISNVTGGQIEAEIAQADYWVKHVSQPVKFVQSIQT 
               
               
                   
               
               
                 LAQAGVNVYLEIGVKPVLLSMGRHCLAEQEAVWLPSLRPHSEPWPEILTSLGKLYEQGLNIDWQTVEAGD 
               
               
                   
               
               
                 RRRKLILPTYPFQRQRYWFNQGSWQTVETESVNPGPDDLNDWLYQVAWTPLDTLPPAPEPSAKLWLILGD 
               
               
                   
               
               
                 RHDHQPIEAQFKNAQRVYLGQSNHFPTNAPWEVSADALDNLFTHVGSQNLAGILYLCPPGEDPEDLDEIQ 
               
               
                   
               
               
                 KQTSGFALQLIQTLYQQKIAVPCWFVTHQSQRVLETDAVTGFAQGGLWGLAQAIALEHPELWGGIIDVDD 
               
               
                   
               
               
                 SLPNFAQICQQRQVQQLAVRHQKLYGAQLKKQPSLPQKNLQIQPQQTYLVTGGLGAIGRKIAQWLAAAGA 
               
               
                   
               
               
                 EKVILVSRRAPAADQQTLPTNAVVYPCDLADAAQVAKLFQTYPHIKGIFHAAGTLADGLLQQQTWQKFQT 
               
               
                   
               
               
                 VAAAKMKGTWHLHRHSQKLDLDFFVLFSSVAGVLGSPGQGNYAAANRGMAAIAQYRQAQGLPALAIHWGP 
               
               
                   
               
               
                 WAEGGMANSLSNQNLAWLPPPQGLTILEKVLGAQGEMGVFKPDWQNLAKQFPEFAKTHYFAAVIPSAEAV 
               
               
                   
               
               
                 PPTASIFDKLINLEASQRADYLLDYLRRSVAQILKLEIEQIQSHDSLLDLGMDSLMIMEAIASLKQDLQL 
               
               
                   
               
               
                 MLYPREIYERPRLDVLTAYLAAEFTKAHDSEAATAAAAIPSQSLSVKTKKQWQKPDHKNPNPIA FILSSP   
               
               
                   
               
               
                 
                   RSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQAKVNQWV 
                 
               
               
                   
               
               
                 
                   KANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTRLRMDKL 
                 
               
               
                   
               
               
                 
                   LGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDEALLNPY 
                 
               
               
                   
               
               
                 SGDRLTDGLHQQSMGVGDPNFLQHKTIDPALADKWRSITLPAALQLDTIQLAETFAYDLPQEPQLTPQTQ 
               
               
                   
               
               
                 SLPSMVER FVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAH   
               
               
                   
               
               
                 
                   AQSYSMLDFLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNH 
                 
               
               
                   
               
               
                 
                   LTTHLDYLAAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEF 
                 
               
               
                   
               
               
                   NGISRRRYLALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIV YQ 
               
               
                   
               
               
                 QLQTPVPKTQGLHHHHHHSAWSHPQFEK 
               
               
                   
               
               
                 SEQ ID NO: 2 
                   
               
               
                   Synechococcus elongatus  NonA (SYNPCC7002_A1173) 
                   
               
               
                 ST domain protein sequence 
               
               
                 FILSSPRSGSTLLRVMLAGHPGLYSPPELHLLPFETMGDRHQELGLSHLGEGLQRALMDLENLTPEASQA 
               
               
                   
               
               
                 KVNQWVKANTPIADIYAYLQRQAEQRLLIDKSPSYGSDRHILDHSEILFDQAKYIHLVRHPYAVIESFTR 
               
               
                   
               
               
                 LRMDKLLGAEQQNPYALAESIWRTSNRNILDLGRTVGADRYLQVIYEDLVRDPRKVLTNICDFLGVDFDE 
               
               
                   
               
               
                 ALLNPY 
               
               
                   
               
               
                 SEQ ID NO: 3 
                   
               
               
                   Synechococcus elongatus  NonA (SYNPCC7002_A1173) 
                   
               
               
                 TE domain protein sequence 
               
               
                 FVTVRGLETCLCEWGDRHQPLVLLLHGILEQGASWQLIAPQLAAQGYWVVAPDLRGHGKSAHAQSYSMLD 
               
               
                   
               
               
                 FLADVDALAKQLGDRPFTLVGHSMGSIIGAMYAGIRQTQVEKLILVETIVPNDIDDAETGNHLTTHLDYL 
               
               
                   
               
               
                 AAPPQHPIFPSLEVAARRLRQATPQLPKDLSAFLTQRSTKSVEKGVQWRWDAFLRTRAGIEFNGISRRRY 
               
               
                   
               
               
                 LALLKDIQAPITLIYGDQSEFNRPADLQAIQAALPQAQRLTVAGGHNLHFENPQAIAQIV