# Patent

## Title
Methods and compositions for the recombinant biosynthesis of n-alkanes

## Abstract

The present disclosure identifies methods and compositions for modifying photoautotrophic organisms as hosts, such that the organisms efficiently convert carbon dioxide and light into n-alkanes, and in particular the use of such organisms for the commercial production of n-alkanes and related molecules.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to earlier filed U.S. Provisional Patent Application No. 61/224,463 filed, Jul. 9, 2009, U.S. Provisional Patent Application No. 61/228,937, filed Jul. 27, 2009, and U.S. utility application Ser. No. 12/759,657, filed Apr. 13, 2010, the disclosures of which are incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
This application contains a computer-readable Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 30, 2010, is named “17078_US_Sequence Listing.txt”, lists 128 sequences, and is 332 kb in size.


FIELD OF THE INVENTION
The present disclosure relates to methods for conferring alkane-producing properties to a heterotrophic or photoautotrophic host, such that the modified host can be used in the commercial production of bioalkanes.
BACKGROUND OF THE INVENTION
Many existing photoautotrophic organisms (i.e., plants, algae, and photosynthetic bacteria) are poorly suited for industrial bioprocessing and have therefore not demonstrated commercial viability. Such organisms typically have slow doubling times (3-72 hrs) compared to industrialized heterotrophic organisms such as Escherichia coli (20 minutes), reflective of low total productivities. While a desire for the efficient biosynthetic production of fuels has led to the development of photosynthetic microorganisms which produce alkyl esters of fatty acids, a need still exists for methods of producing hydrocarbons, e.g., alkanes, using photosynthetic organisms.
SUMMARY OF THE INVENTION
The present invention provides, in certain embodiments, isolated polynucleotides comprising or consisting of nucleic acid sequences selected from the group consisting of the coding sequences for AAR and ADM enzymes, nucleic acid sequences that are codon-optimized variants of these sequences, and related nucleic acid sequences and fragments.
An AAR enzyme refers to an enzyme with the amino acid sequence of the SYNPCC7942—1594 protein (SEQ ID NO: 6) or a homolog thereof, wherein a SYNPCC7942—1594 homolog is a protein whose BLAST alignment (i) covers >90% length of SYNPCC7942—1594, (ii) covers >90% of the length of the matching protein, and (iii) has >50% identity with SYNPCC7942—1594 (when optimally aligned using the parameters provided herein), and retains the functional activity of SYNPCC7942—1594, i.e., the conversion of an acyl-ACP (ACP=acyl carrier protein) to an alkanal. An ADM enzyme refers to an enzyme with the amino acid sequence of the SYNPCC7942—1593 protein (SEQ ID NO: 8) or a homolog thereof, wherein a SYNPCC7942—1593 homolog is defined as a protein whose amino acid sequence alignment (i) covers >90% length of SYNPCC7942—1593, (ii) covers >90% of the length of the matching protein, and (iii) has >50% identity with SYNPCC7942—1593 (when aligned using the preferred parameters provided herein), and retains the functional activity of SYNPCC7942—1593, i.e., the conversion of an n-alkanal to an (n−1)-alkane. Exemplary AAR and ADM enzymes are listed in Table 1 and Table 2, respectively. Genes encoding AAR or ADM enzymes are referred to herein as AAR genes (aar) or ADM genes (adm), respectively.
Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: none; Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Maximum alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
While Applicants refer herein to an alkanal decarboxylative monooxygenase enzyme, Applicants do so without intending to be bound to any particular reaction mechanism unless expressly set forth. For example, whether the enzyme encoded by SYNPCC7942—1593 or any other ADM gene carries out a decarbonylase or a decarboxylase reaction does not affect the utility of Applicants' invention, unless expressly set forth herein to the contrary.
The present invention further provides isolated polypeptides comprising or consisting of polypeptide sequences selected from the group consisting of the sequences listed in Table 1 and Table 2, and related polypeptide sequences, fragments and fusions. Antibodies that specifically bind to the isolated polypeptides of the present invention are also contemplated.
The present invention also provides methods for expressing a heterologous nucleic acid sequence encoding AAR and ADM in a host cell lacking catalytic activity for AAR and ADM (thereby conferring n-alkane producing capability in the host cell), or for expressing a nucleic acid encoding AAR and ADM in a host cell which comprises native AAR and/or ADM activity (thereby enhancing n-alkane producing capability in the host cell).
In addition, the present invention provides methods for producing carbon-based products of interest using the AAR and ADM genes, proteins and host cells described herein. For example, in one embodiment the invention provides a method for producing hydrocarbons, comprising: (i) culturing an engineered cyanobacterium in a culture medium, wherein said engineered cyanobacterium comprises a recombinant AAR enzyme and a recombinant ADM enzyme; and (ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered cynanobacterium into n-alkanes, wherein at least one of said n-alkanes is selected from the group consisting of n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, and n-heptadecane, and wherein the amount of said n-alkanes produced is between 0.1% and 5% dry cell weight and at least two times the amount produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking said recombinant AAR and ADM enzymes.
In a related embodiment, the amount on n-alkanes produced by the engineered cyanobacterium is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% DCW, and at least two times the amount produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking said recombinant AAR and ADM enzymes.
In a related embodiment, at least one of said recombinant enzymes is heterologous with respect to said engineered cyanobacterium. In another embodiment, said cyanobacterium does not synthesize alkanes in the absence of the expression of one or both of the recombinant enzymes. In another embodiment, at least one of said recombinant AAR or ADM enzymes is not heterologous to said engineered cyanobacterium.
In another related embodiment of the method, said engineered cyanobacterium further produces at least one n-alkene or n-alkanol. In yet another embodiment, the engineered cyanobacterium produces at least one n-alkene or n-alkanol selected from the group consisting of n-pentadecene, n-heptadecene, and 1-octadecanol. In a related embodiment, said n-alkanes comprise predominantly n-heptadecane, n-pentadecane or a combination thereof. In a related embodiment, more n-heptadecane and/or n-pentadecane are produced than all other n-alkane products combined. In yet another related embodiment, more n-heptadecane and/or n-pentadecane are produced by the engineered cyanobacterium than any other n-alkane or n-alkene produced by the engineered cyanobacterium. In yet another related embodiment, at least one n-pentadecene produced by said engineered cyanobacterium is selected from the group consisting of cis-3-heptadecene and cis-4-pentadecene. In yet another related embodiment, at least one n-heptadecene produced by said engineered cyanobacterium is selected from the group consisting of cis-4-pentadecene, cis-6-heptadecene, cis-8-heptadecene, cis-9-heptadecene, and cis, cis-heptadec-di-ene.
In yet another related embodiment, the invention further provides a step of isolating at least one n-alkane, n-alkene or n-alkanol from said engineered cyanobacterium or said culture medium. In yet another related embodiment, the engineered cyanobacterium is cultured in a liquid medium. In yet another related embodiment, the engineered cyanobacterium is cultured in a photobioreactor.
In another related embodiment, the AAR and/or ADM enzymes are encoded by a plasmid. In yet another related embodiment, the AAR and/or ADM enzymes are encoded by recombinant genes incorporated into the genome of the engineered cyanobacterium. In yet another related embodiment, the AAR and/or ADM enzymes are encoded by genes which are present in multiple copies in said engineered cyanobacterium. In yet another related embodiment, the recombinant AAR and/or ADM enzymes are encoded by genes which are part of an operon, wherein the expression of said genes is controlled by a single promoter. In yet another related embodiment, the recombinant AAR and/or ADM enzymes are encoded by genes which are expressed independently under the control of separate promoters. In yet another related embodiment, expression of the recombinant AAR and/or ADM enzymes in an engineered cyanobacterium is controlled by a promoter selected from the group consisting of a cI promoter, a cpcB promoter, a lacI-trc promoter, an EM7 promoter, an aphII promoter, a nirA promoter, and a nir07 promoter (referred to herein as “P(nir07)”). In yet another related embodiment, the enzymes are encoded by genes which are part of an operon, wherein the expression of said genes is controlled by one or more inducible promoters. In yet another related embodiment, at least one promoter is a urea-repressible, nitrate-inducible promoter. In yet another related embodiment, the urea-repressible, nitrate-inducible promoter is a nirA-type promoter. In yet another related embodiment, the nirA-type promoter is P(nir07) (SEQ ID NO: 24).
In yet another related embodiment, the cyanobacterium species that is engineered to express recombinant AAR and/or ADM enzymes produces less than approximately 0.01% DCW n-heptadecane or n-pentadecane in the absence of said recombinant AAR and/or ADM enzymes, 0.01% DCW corresponding approximately to the limit of detection of n-heptadecane and n-pentadecane by the gas chromatographic/flame ionization detection methods described herein. In another related embodiment, the engineered cyanobacterium of the method is a thermophile. In yet another related embodiment, the engineered cyanobacterium of the method is selected from the group consisting of an engineered Synechococcus sp. PCC7002 and an engineered Thermosynechococcus elongatus BP-1.
In yet another related embodiment, the recombinant AAR and/or ADM enzymes are selected from the group of enzymes listed in Table 1 and Table 2, respectively. In yet another related embodiment, the recombinant AAR enzymes are selected from the group consisting of SYNPCC7942—1594, tll1312, PMT9312—0533, and cce—1430. In yet another related embodiment, the recombinant ADM enzymes are selected from the group consisting of SYNPCC7942—1593, tll1313, PMT9312—0532, and cce—0778.
In yet another related embodiment, the recombinant AAR and ADM enzymes have the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:12, respectively. In certain embodiments, the recombinant AAR and ADM enzymes are encoded by SEQ ID NOs: 9 and 11, respectively. In yet other embodiments, the recombinant AAR and ADM enzymes are encoded by SEQ ID NOs: 26 and 28, respectively, or SEQ ID NOs: 30 and 31 respectively, and have the amino acid sequences of SEQ ID NOs: 27 and 28, respectively. In certain embodiments, the recombinant AAR and ADM enzymes are encoded by SEQ ID NOs: 1 and 3, respectively, and have the amino acid sequences of SEQ NOs: 2 and 4, respectively. In still other embodiments, the recombinant AAR and ADM enzymes are encoded by SEQ ID NOs: 5 and 7, respectively, and have the amino acid sequences of SEQ ID NOs: 6 and 8, respectively.
In yet another related embodiment, the method comprising culturing the engineered cyanobacterium in the presence of an antibiotic, wherein said antibiotic selects for the presence of a recombinant gene encoding an AAR and/or ADM enzyme. In certain embodiments, the antibiotic is spectinomycin or kanamycin. In related embodiments, the amount of spectinomycin in the culture media is between 100 and 700 μg/ml, e.g., 100, 200, 300, 400, 500, 600, or 700 μg/ml of spectinomycin can be added to the culture media. In certain embodiments, the amount of spectinomycin added is about 600 μg/ml, and the amount of n-alkanes produced by the engineered cyanobacterium is at least about 3%, 4% or 5% DCW.
In another embodiment, the method for producing hydrocarbons comprises culturing a cyanobacterium expressing recombinant AAR and/or ADM enzymes in the presence of an exogenous substrate for one or both enzymes. In a related embodiment, the substrate is selected from the group consisting of an acyl-ACP, an acyl-CoA, and a fatty aldehyde. In another related embodiment, exogenous fatty alcohols or fatty esters or other indirect substrates can be added and converted to acyl-ACP or acyl-CoA by the cyanobacterium.
In yet another embodiment, the invention provides a composition comprising an n-alkane produced by any of the recombinant biosynthetic methods described herein. In yet another embodiment, the invention provides a composition comprising an n-alkene or n-alkanol produced by any of the recombinant biosynthetic methods described herein.
In certain embodiments, the invention provides an engineered host cell for producing an n-alkane, wherein said cell comprises one or more recombinant protein activities selected from the group consisting of an acyl-CoA reductase activity, an acyl-ACP reductase activity, an alkanal decarboxylative monooxygenase activity, and an electron donor activity. In related embodiments, the host cell comprises a recombinant acyl-ACP reductase activity, a recombinant alkanal decarboxylative monooxygenase activity, and a recombinant electron donor activity. In other embodiments, the host cell comprises a recombinant acyl-ACP reductase activity and a recombinant alkanal decarboxylative monooxygenase activity. In certain embodiments, the electron donor activity is a ferredoxin. In certain related embodiments, the host cell is capable of photosynthesis. In still other related embodiments, the host cell is a cyanobacterium. In still other embodiments, the host cell is a gram-negative bacterium, a gram-positive bacterium, or a yeast species.
In other embodiments, the invention provides an isolated or recombinant polynucleotide comprising or consisting of a nucleic acid sequence selected from the group consisting of: (a) SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 14, 30 or 31; (b) a nucleic acid sequence that is a degenerate variant of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 30 or 31; (c) a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 14, 30 or 31; (d) a nucleic acid sequence that encodes a polypeptide having the amino kid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 27 or 29; (e) a nucleic acid sequence that encodes a polypeptide at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO:2, 4, 6, 8, 10, 12, 27 or 39; and (f) a nucleic acid sequence that hybridizes under stringent conditions to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 30 or 31. In related embodiments, the nucleic acid sequence encodes a polypeptide having acyl-ACP reductase activity or alkanal decarboxylative monooxygenase activity.
In yet another embodiment, the invention provides an isolated, soluble polypeptide with alkanal decarboxylative monooxygenase activity wherein, in certain related embodiments, the polypeptide has an amino acid sequence of one of the proteins listed in Table 2. In related embodiments, the polypeptide has the amino acid sequence identical to, or at least 95% identical to, SEQ ID NO: 4, 8, 12 or 29.
In yet another embodiment, the invention provides a method for synthesizing an n-alkane from an acyl-ACP in vitro, comprising: contacting an acyl-ACP with a recombinant acyl-ACP reductase, wherein said acyl-ACP reductase converts said acyl-ACP to an n-alkanal; then contacting said n-alkanal with a recombinant, soluble alkanal decarboxylative monooxygenase in the presence of an electron donor, wherein said alkanal decarboxylative monooxygenase converts said n-alkanal to an (n−1) alkane. In a related embodiment, the invention provides a method for synthesizing an n-alkane from an n-alkanal in vitro, comprising: contacting said n-alkanal with a recombinant, soluble alkanal decarboxylative monooxygenase in the presence of an electron donor, wherein said alkanal decarboxylative monooxygenase converts said n-alkanal to an (n−1)-alkane. In certain related embodiments, the electron donor is a ferredoxin protein.
In another embodiment, the invention provides engineered cyanobacterial cells comprising recombinant AAR and ADM enzymes, wherein said cells comprise between 0.1% and 5%, between 1% and 5%, or between 2% and 5% dry cell weight n-alkanes, wherein said n-alkanes are predominantly n-pentadecane, n-heptadecane, or a combination thereof.
In other embodiments, the invention provides one of the expression and/or transformation vectors disclosed herein. In other related embodiments, the invention provides methods of using one of the expression and/or transformation vectors disclosed herein to transform a microorganism, e.g., a cyanobacterium.
In yet another embodiment of the method for producing hydrocarbons, the AAR and ADM enzymes are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6 and SEQ ID NO: 8, respectively. In a related embodiment, the engineered cyanobacterium produces n-pentadecane and n-heptadecane, wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 20%. In yet another related embodiment, the engineered cyanobacterium produces n-pentadecane and n-heptadecane, wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is less than 30%. In yet another related embodiment, the engineered cyanobacterium produces n-pentadecane and n-heptadecane, wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is between 20% and 30%.
In yet another embodiment of the method for producing hydrocarbons, the AAR and ADM enzymes are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:10 and SEQ ID NO: 12, respectively. In a related embodiment, the engineered cyanobacterium produces n-pentadecane and n-heptadecane, wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 50%. In yet another related embodiment, the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is less than 60%. In yet another related embodiment, the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is between 50% and 60%.
In yet another embodiment of the method for producing hydrocarbons, the engineered cyanobacterium comprises at least two distinct recombinant ADM enzymes and at least two distinct recombinant AAR enzymes. In a related embodiment, said engineered cyanobacterium comprises at least one operon encoding AAR and ADM enzymes which are at least 95% identical to SEQ ID NO: 27 and SEQ ID NO: 29, respectively. In yet another related embodiment, said engineered cyanobacterium comprises at least one operon encoding AAR and ADM enzymes which are at least 95% identical to SEQ ID NO:10 and SEQ ID NO: 12, respectively. In yet another related embodiment, expression of said AAR and ADM enzymes is controlled by an inducible promoter, e.g., a P(nir07) promoter. In yet another related embodiment, said recombinant ADM and AAR enzymes are chromosomally integrated. In yet another related embodiment, said engineered cyanobacterium produces n-alkanes in the presence of an inducer, and wherein at least 95% of said n-alkanes are n-pentadecane and n-heptadecane, and wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 80%.
In yet another embodiment of the method for producing hydrocarbons, the engineered cyanobacterium comprises recombinant AAR and ADM enzymes which are at least 95% identical to SEQ ID NO:10 and SEQ ID NO: 12, respectively. In a related embodiment, the recombinant AAR and ADM enzymes are under the control of an inducible promoter, e.g., a P(nir07) promoter. In yet another related embodiment, the engineered cyanobacterium produces at least 0.5% DCW n-alkanes in the presence of an inducer, and wherein said n-alkanes comprise n-pentadecane and n-heptadecane, and wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 50%.
In yet another embodiment, the invention provides a method for modulating the relative amounts of n-pentadecane and n-heptadecane in an engineered cyanobacterium, comprising controlling the expression of one or more recombinant AAR and/or ADM enzymes in said cyanobacterium, wherein said AAR and/or ADM enzymes are at least 95% identical or identical to the AAR and ADM enzymes of SEQ ID NO:s 10, 12, 27 or 29.
In another embodiment, the invention provides an engineered cyanobacterium, wherein said engineered cyanobacterium comprises one or more recombinant genes encoding an AAR enzyme, an ADM enzyme, or both enzymes, wherein at least one of said recombinant genes is under the control of a nitrate-inducible promoter.
In yet another embodiment, the invention provides a recombinant gene, wherein said gene comprises a promoter for controlling expression of said gene, wherein said promoter comprises a contiguous nucleic acid sequence identical to SEQ ID NO: 24.
In yet another embodiment, the invention provides an isolated DNA molecule comprising a promoter, wherein said promoter comprises a contiguous nucleic acid sequence identical to SEQ ID NO: 24.
In yet another embodiment, the invention provides an engineered bacterial strain selected from the group consisting of JCC1469, JCC1281, JCC1683, JCC1685, JCC1076, JCC1170, JCC1221, JCC879 and JCC1084t.
These and other embodiments of the invention are further described in the Figures, Description, Examples and Claims, herein.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts, in panel A, an enzymatic pathway for the production of n-alkanes based on the sequential activity of (1) an AAR enzyme (e.g., tll1312); and (2) an ADM enzyme (e.g., tll1313); B, Biosynthesis of n-alkanal via acyl-CoA. Acyl-CoAs are typically intermediates of fatty acid degradation; C, Biosynthesis of n-alkanal via acyl-ACP. Acyl-ACP's are typically intermediates of fatty acid biosynthesis. Note the three different types of ACP reductase: (i) β-ketoacyl-ACP reductase, (ii) enoyl-ACP reductase, and (iii) acyl-ACP reductase. Acyl-ACP reductase, a new enzyme, generates the substrate for alkanal decarboxylative monooxygenase. CoA, coenzyme A; ACP, acyl carrier protein; D, an alternative acyl-CoA-mediated alkane biosynthetic pathway. See additional discussion in Example 1, herein.
FIG. 2 represents 0-to-2700000-count total ion chromatograms of JCC9a and JCC1076 BHT (butylated hydroxytoluene)-acetone cell pellet extracts, as well as n-alkane and n−1-alkanol authentic standards. Peaks assigned by Method 1 are identified in regular font, those by Method 2 in italic font.
FIG. 3 depicts MS fragmentation spectra of JCC1076 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, n-pentadecane; B, 1-tetradecanol; C, n-heptadecane; D, 1-hexadecanol.
FIG. 4A represents 0-to-7500000-count total ion chromatograms for the BHT-acetone extracts of JCC1113 and JCC1114 cell pellets, as well as C13-C20 n-alkane and C14, C16, and C18 n−1-alkanol authentic standards; B, represents 0-to-720000-count total ion chromatograms for BHT-acetone extracts of JCC1113 and JCC1114 cell pellets, as well as the n-alkane and n-alkanol authentic standards mentioned in 4A.
FIG. 5 depicts MS fragmentation spectra of JCC1113 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, n-tridecane; B, n-tetradecane; C, n-pentadecane; D, n-hexadecane; E, n-heptadecane; F, 1-hexadecanol.
FIG. 6 represents 0-to-6100000-count total ion chromatograms of JCC1170 and JCC1169 BHT-acetone cell pellet extracts versus those of the control strains JCC1113 and JCC1114. No hydrocarbon products were observed in JCC1169. The unidentified peak in JCC1170 is likely cis-11-octadecenal.
FIG. 7 depicts MS fragmentation spectra of JCC1170 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, 1-tetradecanol; B, 1-hexadecanol.
FIG. 8A represents 0-to-75000000-count total ion chromatograms for BHT-acetone extracts of JCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160 and JCC879 cell pellets, as well as C13-C20 n-alkane and C14, C16, and C18 n-alkanol authentic standards. The doublet around 18.0 minutes corresponds to nonadec-di-ene and 1-nonadecene, respectively (data not shown), n-alkenes that are naturally produced by JCC138; 8B represents 0-to-2250000-count total ion chromatograms for BHT-acetone extracts of JCC1221 and JCC879 cell pellets, as well as the n-alkane and n-alkanol authentic standards mentioned in 8A.
FIG. 9 depicts MS fragmentation spectra of JCC1221 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, n-tridecane; B, n-tetradecane; C, n-pentadecane; D, n-hexadecane; E, n-heptadecane; F, 1-octadecanol.
FIG. 10 depicts intracellular n-alkane production as a function of spectinomycin concentration in JCC1221.
FIG. 11 represents 0-to-1080000-count total ion chromatograms of the JCC1281 BHT-acetone cell pellet extractant versus that of the control strain JCC138, as well as of authentic standard n-alkanes.
FIG. 12 depicts MS fragmentation spectra of JCC1281 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, n-pentadecane; B, n-heptadecane.
FIG. 13 depicts MS fragmentation spectra of JCC3 peaks assigned by Method 1 (top mass spectrum of each panel), plotted against their respective NIST library hits (bottom mass spectrum of each panel). A, n-pentadecane; B, n-hexadecane; C, n-heptadecane.
FIG. 14 depicts enhanced intracellular production of n-alkanes in JCC1084t compared to the control strain JCC1084. Error bars represent standard deviation of three independent observations.
FIG. 15 represents 0-to-31500000-count total ion chromatograms of JCC1113 and JCC1221 BHT-acetone cell pellet extracts, as well as authentic n-alkane standards.



DETAILED DESCRIPTION OF THE INVENTION
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).
All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties.
The following terms, unless otherwise indicated, shall be understood to have the following meanings:
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.
Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.
As used herein, an “isolated” organic molecule (e.g., an alkane, alkene, or alkanal) is one which is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured. The term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.
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.
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 host cell or progeny thereof) or exogenous (originating from a different host 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 host 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 sequence's that naturally flank it.
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 host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm, is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
The nucleic acids (also referred to as polynucleotides) of this 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.
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)).
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.
Deletion: 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.
Knock-out: A gene whose level of expression or activity has been reduced to zero. 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.
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 generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. 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”).
“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.
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.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which 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 “host cell” as used herein. A recombinant host 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.
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.
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.
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.
The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or 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.
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 125I, 32P, 35S, and 3H, 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).
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 comprises 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 the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein 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.
As used herein, the term “antibody” refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives.
Fragments within the scope of the term “antibody” include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab′, Fv, F(ab′).sub.2, and single chain Fv (scFv) fragments.
Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Intracellular Antibodies: Research and Disease Applications, (Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure of which is incorporated herein by reference in its entirety).
As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.
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 the present invention.
A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein.
A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild-type protein.
In an even more preferred embodiment, a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9% overall sequence identity.
Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.
Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.
As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention.
A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.
When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (herein incorporated by reference).
The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.
“Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10−7 M or stronger (e.g., about 10−8 M, 10−9 M or even stronger).
“Percent dry cell weight” refers to a measurement of hydrocarbon production obtained as follows: a defined volume of culture is centrifuged to pellet the cells. Cells are washed then dewetted by at least one cycle of microcentrifugation and aspiration. Cell pellets are lyophilized overnight, and the tube containing the dry cell mass is weighed again such that the mass of the cell pellet can be calculated within ±0.1 mg. At the same time cells are processed for dry cell weight determination, a second sample of the culture in question is harvested, washed, and dewetted. The resulting cell pellet, corresponding to 1-3 mg of dry cell weight, is then extracted by vortexing in approximately 1 ml acetone plus butylated hydroxytolune (BHT) as antioxidant and an internal standard, e.g., n-heptacosane. Cell debris is then pelleted by centrifugation and the supernatant (extractant) is taken for analysis by GC. For accurate quantitation of n-alkanes, flame ionization detection (FID) was used as opposed to MS total ion count. n-Alkane concentrations in the biological extracts were calculated using calibration relationships between GC-FID peak area and known concentrations of authentic n-alkane standards. Knowing the volume of the extractant, the resulting concentrations of the n-alkane species in the extracant, and the dry cell weight of the cell pellet extracted, the percentage of dry cell weight that comprised n-alkanes can be determined.
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.
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, and a cytoplasmic domain.
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.
“Carbon-based Products of Interest” include alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate, 1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3-hydroxypropionic acid (HPA), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; pharmaceuticals and pharmaceutical intermediates such as 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids and other such suitable products of interest. Such products are useful in the context of biofuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, neutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals.
Biofuel: A biofuel refers to any fuel that derives from a biological source. Biofuel can refer to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.
Hydrocarbon: 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.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention pertains. 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.
Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, 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
Alkanes, also known as paraffins, are chemical compounds that consist only of the elements carbon (C) and hydrogen (H) (i.e., hydrocarbons), wherein these atoms are linked together exclusively by single bonds (i.e., they are saturated compounds) without any cyclic structure. n-Alkanes are linear, i.e., unbranched, alkanes. Together, AAR and ADM enzymes function to synthesize n-alkanes from acyl-ACP molecules.
Accordingly, the present invention provides isolated nucleic acid molecules for genes encoding AAR and ADM enzymes, and variants thereof. Exemplary full-length nucleic acid sequences for genes encoding AAR are presented as SEQ ID NOs: 1, 5, and 13, and the corresponding amino acid sequences are presented as SEQ ID NOs: 2, 6, and 10, respectively. Exemplary full-length nucleic acid sequences for genes encoding ADM are presented as SEQ ID NOs: 3, 7, 14, and the corresponding amino acid sequences are presented as SEQ ID NOs: 4, 8, and 12, respectively. Additional nucleic acids provided by the invention include any of the genes encoding the AAR and ADM enzymes in Table 1 and Table 2, respectively.
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 AAR and ADM, and homologs, variants and derivatives thereof expressed in a host cell of interest. The present invention also provides a nucleic acid molecule comprising or consisting of a sequence which is a codon-optimized version of the AAR and ADM genes described herein (e.g., SEQ ID NO: 9 and SEQ ID NO: 11, which are optimized for the expression of the AAR and ADM genes of Prochlorococcus marinus MIT 9312 in Synechoccocus sp. PCC 7002). 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 AAR or ADM gene having at least 76% identity to the 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 another embodiment, the nucleic acid molecule of the present invention encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10 or 12. Preferably, the nucleic acid molecule of the present invention encodes a polypeptide sequence of at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to SEQ ID NO:2, 4, 6, 8, 10 or 12 and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher.
The present 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 (Tm) for the specific DNA hybrid under a particular set of conditions, where the Tm 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 Tm, for the specific DNA hybrid under a particular set of conditions.
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.
The nucleic acid sequence fragments of the present invention display utility in a variety of systems and methods. For example, the fragments may be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be either DNA or RNA. The target nucleic acid sequences may be fractionated (e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ. One of skill in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by Southern blotting) and in measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled, so that their specific hydridization to target sequences can be detected and optionally quantified. One of skill in the art will appreciate that the nucleic acid fragments of the present invention may be used in a wide variety of blotting techniques not specifically described herein.
It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties. Analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, is a well-established utility for sequence fragments in the field of cell and molecular biology. Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosure of each of which is incorporated herein by reference in its entirety.
As is well known in the art, enzyme activities can be measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically (Grubmeyer et al., (1993) 1 Biol. Chem. 268:20299-20304). Alternatively, the activity of the enzyme can be followed using chromatographic techniques, such as by high performance liquid chromatography (Chung and Sloan, (1986) J. Chromatogr. 371:71-81). 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 (1997)Am. Chem. Soc. Symp. Series, 666: 172-208), titration for determining free fatty acids (Komers (1997) Fett/Lipid, 99(2): 52-54), enzymatic methods (Bailer (1991) 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 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.
Vectors
Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the present invention, as described further herein. In a first embodiment, the vectors include the isolated nucleic acid molecules described above. In an alternative embodiment, the vectors of the present invention 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 AAR and/or ADM polypeptide contributing to n-alkane producing activity by a host cell.
Vectors useful for expression of nucleic acids in prokaryotes are well known in the art.
Isolated Polypeptides
According to another aspect of the present invention, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the present invention are provided. In one embodiment, the isolated polypeptide comprises the polypeptide sequence corresponding to SEQ ID NO:2, 4, 6, 8 10 or 12. In an alternative embodiment of the present invention, the isolated polypeptide comprises a polypeptide sequence at least 85% identical to SEQ ID NO:2, 4, 6, 8, 10 or 12. Preferably the isolated polypeptide of the present invention has at least 50%, 60, 70%, 80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NO:2, 4, 6, 8, 10 or 12.
According to other embodiments of the present invention, 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.
The polypeptides 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
In another aspect of the present invention, host cells transformed with the nucleic acid molecules or vectors of the present invention, and descendants thereof, are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences 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.
In a preferred embodiment, the host cell comprises one or more AAR or ADM encoding nucleic acids which express AAR or ADM in the host cell.
In an alternative embodiment, the host cells of the present invention can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present invention so that the activity of the AAR and/or ADM 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
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.
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.
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., >1 g) hypogravity (e.g., <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>9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, which cannot tolerate O2 such as Methanococcus jannaschii; microaerophils, which tolerate some O2 such as Clostridium and aerobes, which require O2 are also contemplated. Gas-tolerant organisms, which tolerate pure CO2 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).
Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea. 
Algae and cyanobacteria include but are not limited to the following genera: 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. A partial list of cyanobacteria that can be engineered to express recombinant AAR and ADM enzymes is also provided in Table 1 and Table 2, herein. Additional cyanobacteria include members of the genus Chamaesiphon, Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira, Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Planktothrix, Prochlorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena, Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia, Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, lyengariella, Nostochopsis, Stigonema and Thermosynechococcus. 
Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium. 
Green sulfur bacteria include but are not limited to the following genera:
Chlorobium, Clathrochloris, and Prosthecochloris. 
Purple sulfur bacteria include but are not limited to the following genera: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis, 
Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira. 
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.
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.
Preferred organisms for the manufacture of n-alkanes according to the methods discloused herein 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, 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).
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.
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. 
A suitable organism for selecting or engineering is autotrophic fixation of CO2 to products. This would cover photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of CO2 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 of prokaryotes. The CO2 fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. See, e.g., Fuchs, G. 1989. Alternative pathways of autotrophic CO2 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 CO2 fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria.
For producing n-alkanes via the recombinant expression of AAR and/or ADM enzymes, an engineered cyanobacteria, e.g., a Synechococcus or Thermosynechococcus species, is preferred. Other preferred organisms include Synechocystis, Klebsiella oxytoca, Escherichia coli or Saccharomyces cerevisiae. Other prokaryotic, archaea and eukaryotic host cells are also encompassed within the scope of the present invention.
Carbon-Based Products of Interest: Hydrocarbons & Alcohols
In various embodiments of the invention, desired hydrocarbons and/or alcohols of certain chain length or a mixture thereof can be produced. In certain aspects, the host cell produces at least one of the following carbon-based products of interest: 1-dodecanol, 1-tetradecanol, 1-pentadecanol, n-tridecane, n-tetradecane, 15:1 n-pentadecane, n-pentadecane, 16:1 n-hexadecene, n-hexadecane, 17:1 n-heptadecene, n-heptadecane, 16:1 n-hexadecen-ol, n-hexadecan-1-ol and n-octadecen-1-ol, as shown in the Examples herein. In other aspects, the carbon chain length ranges from C10 to C20. Accordingly, the invention provides production of various chain lengths of alkanes, alkenes and alkanols suitable for use as fuels & chemicals.
In preferred aspects, the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. These carbon-based products of interest are secreted directly into the medium. Since the invention enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the invention, therefore, can be used directly or used with minimal processing.
Fuel Compositions
In various embodiments, compositions produced by the methods of the invention are used as fuels. Such fuels comply with ASTM standards, for instance, standard specifications for diesel fuel oils D 975-09b, and Jet A, Jet A-1 and Jet B as specified in ASTM Specification D. 1655-68. Fuel compositions may require blending of several products to produce a uniform product. The blending process is relatively straightforward, but the determination of the amount of each component to include in a blend is much more difficult. Fuel compositions may, therefore, include aromatic and/or branched hydrocarbons, for instance, 75% saturated and 25% aromatic, wherein some of the saturated hydrocarbons are branched and some are cyclic. Preferably, the methods of the invention produce an array of hydrocarbons, such as C13-C17 or C10-C15 to alter cloud point. Furthermore, the compositions may comprise fuel additives, which are used to enhance the performance of a fuel or engine. For example, fuel additives can be used to alter the freezing/gelling point, cloud point, lubricity, viscosity, oxidative stability, ignition quality, octane level, and flash point. Fuels compositions may also comprise, among others, antioxidants, static dissipater, corrosion inhibitor, icing inhibitor, biocide, metal deactivator and thermal stability improver.
In addition to many environmental advantages of the invention such as CO2 conversion and renewable source, other advantages of the fuel compositions disclosed herein include low sulfur content, low emissions, being free or substantially free of alcohol and having high cetane number.
Carbon Fingerprinting
Biologically-produced carbon-based products, e.g., ethanol, fatty acids, alkanes, isoprenoids, represent a new commodity for fuels, such as alcohols, diesel and gasoline. Such biofuels have not been produced using biomass but use CO2 as its carbon source. These new fuels may be distinguishable from fuels derived form petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Such products, derivatives, and mixtures thereof may be completely distinguished from their petrochemical derived counterparts on the basis of 14C (fM) and dual carbon-isotopic fingerprinting, indicating new compositions of matter.
There are three naturally occurring isotopes of carbon: 12C, 13C, and 14C. These isotopes occur in above-ground total carbon at fractions of 0.989, 0.011, and 10−12, respectively. The isotopes 12C and 13C are stable, while 14C decays naturally to 14N, a beta particle, and an anti-neutrino in a process with a half-life of 5730 years. The isotope 14C originates in the atmosphere, due primarily to neutron bombardment of 14N caused ultimately by cosmic radiation. Because of its relatively short half-life (in geologic terms), 14C occurs at extremely low levels in fossil carbon. Over the course of 1 million years without exposure to the atmosphere, just 1 part in 1050 will remain 14C.
The 13C:12C ratio varies slightly but measurably among natural carbon sources. Generally these differences are expressed as deviations from the 13C:12C ratio in a standard material. The international standard for carbon is Pee Dee Belemnite, a form of limestone found in South Carolina, with a 13C fraction of 0.0112372. For a carbon source a, the deviation of the 13C:12C ratio from that of Pee Dee Belemnite is expressed as: δa=(Ra/Rs)−1, where Ra=13C:12C ratio in the natural source, and Rs=13C:12C ratio in Pee Dee Belemnite, the standard. For convenience, δa is expressed in parts per thousand, or ‰. A negative value of δa, shows a bias toward 12C over 13C as compared to Pee Dee Belemnite. Table A shows δa and 14C fraction for several natural sources of carbon.







TABLE A







13C:12C variations in natural carbon sources











Source
−δa (‰)
References















Underground coal
32.5
Farquhar et al. (1989) Plant





Mol. Biol., 40: 503-37



Fossil fuels
26
Farquhar et al. (1989) Plant





Mol. Biol., 40: 503-37



Ocean DIC*
  0-1.5
Goericke et al. (1994) Chapter





9 in Stable Isotopes in Ecology





and Environmental Science,





by K. Lajtha and R. H.





Michener, Blackwell





Publishing;





Ivlev (2010) Separation Sci.





Technol. 36: 1819-1914



Atmospheric
6-8
Ivlev (2010) Separation Sci.



CO2

Technol. 36: 1819-1914;





Farquhar et al. (1989) Plant





Mol. Biol., 40: 503-37



Freshwater DIC*
 6-14
Dettman et al. (1999)





Geochim. Cosmochim. Acta





63: 1049-1057



Pee Dee
0
Ivlev (2010) Separation Sci.



Belemnite

Technol. 36: 1819-1914







*DIC = dissolved inorganic carbon






Biological processes often discriminate among carbon isotopes. The natural abundance of 14C is very small, and hence discrimination for or against 14C is difficult to measure. Biological discrimination between 13C and 12C, however, is well-documented. For a biological product p, we can define similar quantities to those above: δp=(Rp/Rs)−1, where Rp=13C:12C ratio in the biological product, and Rs=13C:12C ratio in Pee Dee Belemnite, the standard. Table B shows measured deviations in the 13C:12C ratio for some biological products.







TABLE B







13C:12C variations in selected biological products










Product
−δp(‰)
−D(‰)*
References





Plant sugar/starch from
18-28
  10-20
Ivlev (2010) Separation


atmospheric CO2


Sci. Technol. 36: 1819-1914


Cyanobacterial biomass from
18-31
16.5-31
Goericke et al. (1994)


marine DIC


Chapter 9 in Stable





Isotopes in Ecology and





Environmental Science,





by K. Lajtha and R. H. Michener,





Blackwell





Publishing;





Sakata et al. (1997)





Geochim. Cosmochim.





Acta, 61: 5379-89


Cyanobacterial lipid from marine
39-40
37.5-40
Sakata et al. (1997)


DIC


Geochim. Cosmochim.





Acta, 61: 5379-89


Algal lipid from marine DIC
17-28
15.5-28
Goericke et al. (1994)





Chapter 9 in Stable





Isotopes in Ecology and





Environmental Science,





by K. Lajtha and R. H. Michener,





Blackwell





Publishing;





Abelseon et al. (1961)





Proc. Natl. Acad. Sci.,





47: 623-32


Algal biomass from freshwater
17-36
  3-30
Marty et al. (2008) Limnol.


DIC


Oceanogr.: Methods 6: 51-63


E. coli lipid from plant sugar
15-27
near 0
Monson et al. (1980) J.





Biol. Chem., 255: 11435-41


Cyanobacterial lipid from fossil
63.5-66  
37.5-40
—


carbon


Cyanobacterial biomass from
42.5-57  
16.5-31
—


fossil carbon





*D = discrimination by a biological process in its utilization of 12C vs. 13C (see text)






Table B introduces a new quantity, D. This is the discrimination by a biological process in its utilization of 12C vs. 13C. We define D as follows: D=(Rp/Ra)−1. This quantity is very similar to δa and δp, except we now compare the biological product directly to the carbon source rather than to a standard. Using D, we can combine the bias effects of a carbon source and a biological process to obtain the bias of the biological product as compared to the standard. Solving for δp, we obtain: δp=(D)(δa)+D+δa, and, because (D)(δa) is generally very small compared to the other terms, δp≈δa+D.
For a biological product having a production process with a known D, we may therefore estimate δp by summing δa and D. We assume that D operates irrespective of the carbon source. This has been done in Table B for cyanobacterial lipid and biomass produced from fossil carbon. As shown in the Table A and Table B, above, cyanobacterial products made from fossil carbon (in the form of, for example, flue gas or other emissions) will have a higher δp than those of comparable biological products made from other sources, distinguishing them on the basis of composition of matter from these other biological products. In addition, any product derived solely from fossil carbon will have a negligible fraction of 14C, while products made from above-ground carbon will have a 14C fraction of approximately 10−12.
Accordingly, in certain aspects, the invention provides various carbon-based products of interest characterized as −δp(‰) of about 63.5 to about 66 and D(‰) of about 37.5 to about 40.
Antibodies
In another aspect, the present invention provides isolated antibodies, including fragments and derivatives thereof that bind specifically to the isolated polypeptides and polypeptide fragments of the present invention or to one or more of the polypeptides encoded by the isolated nucleic acids of the present invention. The antibodies of the present invention may be specific for linear epitopes, discontinuous epitopes or conformational epitopes of such polypeptides or polypeptide fragments, either as present on the polypeptide in its native conformation or, in some cases, as present on the polypeptides as denatured, as, e.g., by solubilization in SDS. Among the useful antibody fragments provided by the instant invention are Fab, Fab′, Fv, F(ab)2, and single chain Fv fragments.
By “bind specifically” and “specific binding” is here intended the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to “recognize” a first molecular species when it can bind specifically to that first molecular species.
As is well known in the art, the degree to which an antibody can discriminate as among molecular species in a mixture will depend, in part, upon the conformational relatedness of the species in the mixture; typically, the antibodies of the present invention will discriminate over adventitious binding to unrelated polypeptides by at least two-fold, more typically by at least 5-fold, typically by more than 10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, and on occasion by more than 500-fold or 1000-fold.
Typically, the affinity or avidity of an antibody (or antibody multimer, as in the case of an IgM pentamer) of the present invention for a polypeptide or polypeptide fragment of the present invention will be at least about 1×10−6 M, typically at least about 5×10−7 M, usefully at least about 1×10−7 M, with affinities and avidities of 1×10−8 M, 5×10−9 M, 1×10−10 M and even stronger proving especially useful.
The isolated antibodies of the present invention may be naturally-occurring forms, such as IgG, IgM, IgD, IgE, and IgA, from any mammalian species. For example, antibodies are usefully obtained from species including rodents-typically mouse, but also rat, guinea pig, and hamster-lagomorphs, typically rabbits, and also larger mammals, such as sheep, goats, cows, and horses. The animal is typically affirmatively immunized, according to standard immunization protocols, with the polypeptide or polypeptide fragment of the present invention.
Virtually all fragments of 8 or more contiguous amino acids of the polypeptides of the present invention may be used effectively as immunogens when conjugated to a carrier, typically a protein such as bovine thyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin, conveniently using a bifunctional linker. Immunogenicity may also be conferred by fusion of the polypeptide and polypeptide fragments of the present invention to other moieties. For example, peptides of the present invention can be produced by solid phase synthesis on a branched polylysine core matrix; these multiple antigenic peptides (MAPs) provide high purity, increased avidity, accurate chemical definition and improved safety in vaccine development. See, e.g., Tam et al., Proc. Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al., J. Biol. Chem. 263, 1719-1725 (1988).
Protocols for immunization are well-established in the art. Such protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant. Antibodies of the present invention may be polyclonal or monoclonal, with polyclonal antibodies having certain advantages in immuno-histochemical detection of the proteins of the present invention and monoclonal antibodies having advantages in identifying and distinguishing particular epitopes of the proteins of the present invention. Following immunization, the antibodies of the present invention may be produced using any art-accepted technique. Host cells for recombinant antibody production—either whole antibodies, antibody fragments, or antibody derivatives—can be prokaryotic or eukaryotic. Prokaryotic hosts are particularly useful for producing phage displayed antibodies, as is well known in the art. Eukaryotic cells, including mammalian, insect, plant and fungal cells are also useful for expression of the antibodies, antibody fragments, and antibody derivatives of the present invention. Antibodies of the present invention can also be prepared by cell free translation.
The isolated antibodies of the present invention, including fragments and derivatives thereof, can usefully be labeled. It is, therefore, another aspect of the present invention to provide labeled antibodies that bind specifically to one or more of the polypeptides and polypeptide fragments of the present invention. The choice of label depends, in part, upon the desired use. In some cases, the antibodies of the present invention may usefully be labeled with an enzyme. Alternatively, the antibodies may be labeled with colloidal gold or with a fluorophore. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention may usefully be labeled with biotin. When the antibodies of the present invention are used, e.g., for Western blotting applications, they may usefully be labeled with radioisotopes, such as 33P, 32P, 35S, 3H and 125I. As would be understood, use of the labels described above is not restricted to any particular application.
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 n-alkanes. An enzymatic process for the production of n-alkanes in, e.g., cyanobacteria is shown in FIG. 1A based on the sequential activity of (1) an AAR enzyme, e.g., tll1312, an acyl-ACP reductase; and (2) an ADM enzyme, e.g., tll1313, a putative alkanal decarboxylative monooxygenase, that uses reduced ferredoxin as electron donor. The AAR activity is distinct from the relatively well characterized acyl-CoA reductase activity exhibited by proteins such as Acr1 from Acinetobacter calcoaceticus (Reiser S and Somerville C (1997) J. Bacteriol. 179:2969-2975). A membranous ADM activity has previously been identified in insect microsomal preparations (Reed J R et al. (1994) Proc. Natl. Acad. Sci. USA 91:10000-10004; Reed J R et al. (1995) Musca domestica. Biochemistry 34:16221-16227).
FIGS. 1B and 1C summarize the names and activities of the enzymes involved in the biosynthesis of n-alkanals. FIG. 1B depicts the relatively well characterized acyl-CoA reductase activity (EC 1.2.1.50) exhibited by proteins such as Acr1 from Acinetobacter calcoaceticus. In FIG. 1C, the two well-known ACP-related reductases that are involved in fatty acid biosynthesis, β-ketoacyl-ACP reductase (EC 1.1.1.100) and enoyl-ACP reductase (EC 1.3.1.9, 1.3.1.10), are contrasted with the acyl-ACP reductase (AAR) (no EC number yet assigned) believed to be involved in the biosynthetic pathway for n-alkanes in cyanobacteria. The key difference between AAR and acyl-CoA reductase (EC 1.2.1.50) is that ACP is the acyl carrier rather than coenzyme A. Supporting this distinction, it has been shown that acyl-CoA reductase Acr1 from Acinetobacter calcoaceticus can only generate alkanals from acyl-CoA and not acyl-ACP (Resier S and Somerville C (1997) J Bacteriol. 179: 2969-2975).
ADM also lacks a presently assigned EC number. An alkanal monooxygenase (EC 1.14.14.3), often referred to as luciferase, is known to catalyze the conversion of n-alkanal to n-alkanoic acid. This activity is distinct from the ADM activity (n-alkanal to (n−1)-alkane) proposed herein, although both use n-alkanal and molecular oxygen as substrates.
Cyanobacterial AAR and ADM homologs for production of n-alkanes. In this example, homologs of cyanobacterial AAR and ADM genes (e.g., homologs of Synechococcus elongatus PCC 7942 SYNPCC7942—1594 and/or SYNPCC7942—1593 protein, respectively) are identified using a BLAST search. These proteins can be expressed in a variety of organisms (bacteria, yeast, plant, etc.) for the purpose of generating and isolating n-alkanes and other desired carbon-based products of interest from the organisms. A search of the non-redundant BLAST protein database revealed counterparts for each protein in other cyanobacteria.
To determine the degree of similarity among homologs of the Synechococcus elongatus PCC 7942 SYNPCC7942—1594 protein, the 341-amino acid protein sequence was queried using BLAST (http://blast.ncbi.nlm.nih.gov/) against the “nr” non-redundant protein database. Homologs were taken as matching proteins whose alignments (i) covered >90% length of SYNPCC7942—1594, (ii) covered >90% of the length of the matching protein, and (iii) had >50% identity with SYNPCC7942—1594 (Table 1).







TABLE 1







Protein homologs of SYNPCC7942_1594 (AAR)













BLAST Score,


Organism
SEQ ID NO:
Homolog accession #
E-value













Synechococcus elongatus
6
(SYNPCC7942_1594)
n/a


PCC 7942


Synechococcus elongatus
23
YP_400611.1
706, 0.0


PCC 7942 [cyanobacteria]


taxid 1140


Synechococcus elongatus
24
YP_170761.1
706, 0.0


PCC 6301 [cyanobacteria]


taxid 269084


Anabaena variabilis ATCC
25
YP_323044.1
538, 4e−151


29413 [cyanobacteria] taxid


240292


Nostoc sp. PCC 7120
26
NP_489324.1
535, 3e−150


[cyanobacteria]


taxid 103690


‘Nostoc azollae’ 0708
27
ZP_03763674.1
533, 1e−149


[cyanobacteria]


taxid 551115


Cyanothece sp. PCC 7425
28
YP_002481152.1
526, 9e−148


[cyanobacteria]


taxid 395961


Nodularia spumigena CCY
29
ZP_01628095.1
521, 3e−146


9414 [cyanobacteria] taxid


313624


Lyngbya sp. PCC 8106
30
ZP_01619574.1
520, 6e−146


[cyanobacteria]


taxid 313612


Nostoc punctiforme PCC
31
YP_001865324.1
520, 7e−146


73102 [cyanobacteria] taxid


63737


Trichodesmium erythraeum
32
YP_721978.1
517, 6e−145


IMS101 [cyanobacteria]


taxid 203124


Thermosynechococcus
2
NP_682102.1
516, 2e−144


elongatus BP-1


[cyanobacteria]


taxid 197221


Acaryochloris marina
33
YP_001518341.1
512, 2e−143


MBIC11017 [cyanobacteria]


taxid 329726


Cyanothece sp. PCC 8802
34
ZP_03142196.1
510, 8e−143


[cyanobacteria]


taxid 395962


Cyanothece sp. PCC 8801
35
YP_002371106.1
510, 8e−143


[cyanobacteria]


taxid 41431


Microcoleus chthonoplastes
36
YP_002619867.1
509, 2e−142


PCC 7420 [cyanobacteria]


taxid 118168


Arthrospira maxima CS-328
37
ZP_03273554.1
507, 7e−142


[cyanobacteria]


taxid 513049


Synechocystis sp. PCC 6803
38
NP_442146.1
504, 5e−141


[cyanobacteria]


taxid 1148


Cyanothece sp. CCY 0110
39
ZP_01728620.1
501, 4e−140


[cyanobacteria]


taxid 391612


Synechococcus sp. PCC 7335
40
YP_002711557.1
500, 1e−139


[cyanobacteria]


taxid 91464


Cyanothece sp. ATCC 51142
41
YP_001802846.1
489, 2e−136


[cyanobacteria]


taxid 43989


Gloeobacter violaceus PCC
42
NP_926091.1
487, 7e−136


7421 [cyanobacteria] taxid


251221


Microcystis aeruginosa
43
YP_001660322.1
486, 1e−135


NIES-843 [cyanobacteria]


taxid 449447


Crocosphaera watsonii WH
44
ZP_00516920.1
486, 1e−135


8501 [cyanobacteria] taxid


165597


Microcystis aeruginosa PCC
45
emb|CAO90781.1
484, 8e−135


7806 [cyanobacteria] taxid


267872


Synechococcus sp. WH 5701
46
ZP_01085337.1
471, 4e−131


[cyanobacteria]


taxid 69042


Synechococcus sp. RCC307
47
YP_001227841.1
464, 8e−129


[cyanobacteria]


taxid 316278


uncultured marine type-A
48
gb|ABD96327.1
462, 2e−128


Synechococcus GOM 3O6


[cyanobacteria]


taxid 364150


Synechococcus sp. WH 8102
49
NP_897828.1
462, 2e−128


[cyanobacteria]


taxid 84588


Synechococcus sp. WH 7803
50
YP_001224378.1
459, 2e−127


[cyanobacteria]


taxid 32051


uncultured marine type-A
51
gb|ABD96480.1
458, 3e−127


Synechococcus GOM 5D20


[cyanobacteria]


taxid 364154


Synechococcus sp. WH 7805
52
ZP_01123215.1
457, 5e−127


[cyanobacteria]


taxid 59931


uncultured marine type-A
53
gb|ABB92249.1
457, 8e−127


Synechococcus 5B2


[cyanobacteria]


taxid 359140


Synechococcus sp. RS9917
54
ZP_01079773.1
456, 2e−126


[cyanobacteria]


taxid 221360


Synechococcus sp. CC9902
55
YP_377636.1
454, 6e−126


[cyanobacteria]


taxid 316279


Prochlorococcus marinus
56
NP_874926.1
453, 9e−126


subsp. marinus str.


CCMP1375 [cyanobacteria]


taxid 167539


Prochlorococcus marinus str.
57
NP_895058.1
453, 1e−125


MIT 9313 [cyanobacteria]


taxid 74547


uncultured marine type-A
58
gb|ABD96274.1
452, 2e−125


Synechococcus GOM 3M9


[cyanobacteria]


taxid 364149


uncultured marine type-A
59
gb|ABD96442.1
452, 2e−125


Synechococcus GOM 4P21


[cyanobacteria]


taxid 364153


Synechococcus sp. BL107
60
ZP_01469469.1
452, 2e−125


[cyanobacteria]


taxid 313625


Cyanobium sp. PCC 7001
61
YP_002597253.1
451, 4e−125


[cyanobacteria]


taxid 180281


Prochlorococcus marinus str.
62
YP_001014416.1
449, 2e−124


NATL1A [cyanobacteria]


taxid 167555


Prochlorococcus marinus str.
63
YP_001010913.1
447, 6e−124


MIT 9515 [cyanobacteria]


taxid 167542


Synechococcus sp. CC9605
64
YP_381056.1
447, 8e−124


[cyanobacteria]


taxid 110662


Prochlorococcus marinus str.
65
YP_001550421.1
446, 2e−123


MIT 9211 [cyanobacteria]


taxid 93059


Prochlorococcus marinus
66
NP_892651.1
446, 2e−123


subsp. pastoris str.


CCMP1986 [cyanobacteria]


taxid 59919


Prochlorococcus marinus str.
67
YP_001090783.1
445, 3e−123


MIT 9301 [cyanobacteria]


taxid 167546


Synechococcus sp. RS9916
68
ZP_01472595.1
445, 3e−123


[cyanobacteria]


taxid 221359


Prochlorococcus marinus str.
69
YP_293055.1
445, 4e−123


NATL2A [cyanobacteria]


taxid 59920


Prochlorococcus marinus str.
70
YP_002673377.1
444, 7e−123


MIT 9202 [cyanobacteria]


taxid 93058


Synechococcus sp. CC9311
71
YP_731192.1
443, 1e−122


[cyanobacteria]


taxid 64471


Prochlorococcus marinus str.
72
YP_001483815.1
442, 2e−122


MIT 9215 [cyanobacteria]


taxid 93060


Prochlorococcus marinus str.
73
YP_001008982.1
442, 3e−122


AS9601 [cyanobacteria]


taxid 146891


Synechococcus sp. JA-3-3Ab
74
YP_473896.1
441, 5e−122


[cyanobacteria]


taxid 321327


Synechococcus sp. JA-2-
75
YP_478638.1
440, 8e−122


3B′a(2-13) [cyanobacteria]


taxid 321332


Prochlorococcus marinus str.
76
YP_397030.1
436, 1e−120


MIT 9312 [cyanobacteria]


taxid 74546









To determine the degree of similarity among homologs of the Synechococcus elongatus PCC 7942 SYNPCC7942—1593 protein, the 231 amino acid protein sequence was queried using BLAST (http://blast.ncbi.nlm.nih.gov/) against the “nr” non-redundant protein database. Homologs were taken as matching proteins whose alignments (i) covered >90% length of SYNPCC7942—1593, (ii) covered >90% of the length of the matching protein, (iii) and had >50% identity with SYNPCC7942—1593 (Table 2).







TABLE 2







Protein homologs of SYNPCC7942_1593 (ADM)













BLAST Score,


Organism
SEQ ID NO:
Homolog accession #
E-value













Synechococcus elongatus PCC
8
(SYNPCC7942_1593)
n/a


7942 [cyanobacteria]


Synechococcus elongatus PCC
77
YP_400610.1
475, 1e−132


7942 [cyanobacteria] taxid 1140


Synechococcus elongatus PCC
78
YP_170760.1
475, 2e−132


6301 [cyanobacteria] taxid 269084


Arthrospira maxima CS-328
79
ZP_03273549.1
378, 3e−103


[cyanobacteria]


taxid 513049


Microcoleus chthonoplastes PCC
80
YP_002619869.1
376, 1e−102


7420 [cyanobacteria]


taxid 118168


Lyngbya sp. PCC 8106
81
ZP_01619575.1
374, 5e−102


[cyanobacteria]


taxid 313612


Nodularia spumigena CCY 9414
82
ZP_01628096.1
369, 1e−100


[cyanobacteria]


taxid 313624


Microcystis aeruginosa NIES-843
83
YP_001660323.1
367, 5e−100


[cyanobacteria] taxid 449447


Microcystis aeruginosa PCC 7806
84
emb|CAO90780.1
364, 3e−99


[cyanobacteria] taxid 267872


Nostoc sp. PCC 7120
85
NP_489323.1
363, 1e−98


[cyanobacteria]


taxid 103690


Anabaena variabilis ATCC 29413
86
YP_323043.1
362, 2e−98


[cyanobacteria] taxid 240292


Crocosphaera watsonii WH 8501
87
ZP_00514700.1
359, 1e−97


[cyanobacteria] taxid 165597


Trichodesmium erythraeum
88
YP_721979.1
358, 2e−97


IMS101 [cyanobacteria] taxid


203124


Synechococcus sp. PCC 7335
89
YP_002711558.1
357, 6e−97


[cyanobacteria]


taxid 91464


‘Nostoc azollae’ 0708
90
ZP_03763673.1
355, 3e−96


[cyanobacteria]


taxid 551115


Synechocystis sp. PCC 6803
91
NP_442147.1
353, 5e−96


[cyanobacteria]


taxid 1148


Cyanothece sp. ATCC 51142
92
YP_001802195.1
352, 2e−95


[cyanobacteria]


taxid 43989


Cyanothece sp. CCY 0110
93
ZP_01728578.1
352, 2e−95


[cyanobacteria]


taxid 391612


Cyanothece sp. PCC 7425
94
YP_002481151.1
350, 7e−95


[cyanobacteria]


taxid 395961


Nostoc punctiforme PCC 73102
95
YP_001865325.1
349, 1e−94


[cyanobacteria]


taxid 63737


Acaryochloris marina
96
YP_001518340.1
344, 4e−93


MBIC11017 [cyanobacteria] taxid


329726


Cyanothece sp. PCC 8802
97
ZP_03142957.1
342, 1e−92


[cyanobacteria]


taxid 395962


Cyanothece sp. PCC 8801
98
YP_002370707.1
342, 1e−92


[cyanobacteria]


taxid 41431


Thermosynechococcus elongatus
4
NP_682103.1
332, 2e−89


BP-1 [cyanobacteria]


taxid 197221


Synechococcus sp. JA-2-3B′a(2-
99
YP_478639.1
319, 1e−85


13) [cyanobacteria] taxid 321332


Synechococcus sp. RCC307
100
YP_001227842.1
319, 1e−85


[cyanobacteria]


taxid 316278


Synechococcus sp. WH 7803
101
YP_001224377.1
313, 8e−84


[cyanobacteria]


taxid 32051


Synechococcus sp. WH 8102
102
NP_897829.1
311, 3e−83


[cyanobacteria]


taxid 84588


Synechococcus sp. WH 7805
103
ZP_01123214.1
310, 6e−83


[cyanobacteria]


taxid 59931


uncultured marine type-A
104
gb|ABD96376.1
309, 1e−82


Synechococcus GOM 3O12


[cyanobacteria]


taxid 364151


Synechococcus sp. JA-3-3Ab
105
YP_473897.1
309, 1e−82


[cyanobacteria]


taxid 321327


uncultured marine type-A
106
gb|ABD96328.1
309, 1e−82


Synechococcus GOM 3O6


[cyanobacteria]


taxid 364150


uncultured marine type-A
107
gb|ABD96275.1
308, 2e−82


Synechococcus GOM 3M9


[cyanobacteria]


taxid 364149


Synechococcus sp. CC9311
108
YP_731193.1
306, 7e−82


[cyanobacteria]


taxid 64471


uncultured marine type-A
109
gb|ABB92250.1
306, 9e−82


Synechococcus 5B2


[cyanobacteria]


taxid 359140


Synechococcus sp. WH 5701
110
ZP_01085338.1
305, 3e−81


[cyanobacteria]


taxid 69042


Gloeobacter violaceus PCC 7421
111
NP_926092.1
303, 8e−81


[cyanobacteria] taxid 251221


Synechococcus sp. RS9916
112
ZP_01472594.1
303, 9e−81


[cyanobacteria]


taxid 221359


Synechococcus sp. RS9917
113
ZP_01079772.1
300, 6e−80


[cyanobacteria]


taxid 221360


Synechococcus sp. CC9605
114
YP_381055.1
300, 7e−80


[cyanobacteria]


taxid 110662


Prochlorococcus marinus str. MIT
115
YP_001016795.1
294, 4e−78


9303 [cyanobacteria]


taxid 59922


Cyanobium sp. PCC 7001
116
YP_002597252.1
294, 6e−78


[cyanobacteria]


taxid 180281


Prochlorococcus marinus str. MIT
117
NP_895059.1
291, 3e−77


9313 [cyanobacteria]


taxid 74547


Synechococcus sp. CC9902
118
YP_377637.1
289, 1e−76


[cyanobacteria]


taxid 316279


Prochlorococcus marinus str. MIT
119
YP_001090782.1
287, 5e−76


9301 [cyanobacteria]


taxid 167546


Synechococcus sp. BL107
120
ZP_01469468.1
287, 6e−76


[cyanobacteria]


taxid 313625


Prochlorococcus marinus str.
121
YP_001008981.1
286, 2e−75


AS9601 [cyanobacteria] taxid


146891


Prochlorococcus marinus str. MIT
12
YP_397029.1
282, 1e−74


9312 [cyanobacteria]


taxid 74546


Prochlorococcus marinus subsp.
122
NP_892650.1
280, 9e−74


pastoris str. CCMP1986


[cyanobacteria]


taxid 59919


Prochlorococcus marinus str. MIT
123
YP_001550420.1
279, 2e−73


9211 [cyanobacteria]


taxid 93059


Prochlorococcus marinus str.
124
YP_293054.1
276, 9e−73


NATL2A [cyanobacteria]


taxid 59920


Prochlorococcus marinus str.
125
YP_001014415.1
276, 9e−73


NATL1A [cyanobacteria]


taxid 167555


Prochlorococcus marinus subsp.
126
NP_874925.1
276, 1e−72


marinus str. CCMP1375


[cyanobacteria]


taxid 167539


Prochlorococcus marinus str. MIT
127
YP_001010912.1
273, 6e−72


9515 [cyanobacteria]


taxid 167542


Prochlorococcus marinus str. MIT
128
YP_001483814.1
273, 9e−72


9215 [cyanobacteria]


taxid 93060









The amino acid sequences referred to in the Table, as those sequences appeared in the NCBI database on Jul. 9, 2009, by accession number are incorporated by reference herein.
An AAR enzyme from Table 1, and/or an ADM enzyme from Table 2, or both can be expressed in a host cell of interest, wherein the host may be a heterologous host or the native host, i.e., the species from which the genes were originally derived. In one embodiment, the invention provides a method of imparting n-alkane synthesis capability in a heterologous organism, lacking native homologs of AAR and/or ADM, by engineering the organism to express a gene encoding one of the enzymes listed in Table 1 or Table 2. Also provided are methods of modulating n-alkane synthesis in an organism which already expresses one or both of the AAR and ADM enzymes by increasing the expression of the native enzymes, or by augmenting native gene expression by the recombinant expression of heterologous AAR and/or ADM enzymes. In addition, the invention provides methods of modulating the degree of alkane synthesis by varying certain parameters, including the identity and/or compatibility of electron donors, culture conditions, promoters for expressing AAR and/or ADM enzymes, and the like.
If the host lacks a suitable electron donor or lacks sufficient levels of a suitable electron donor to achieve production of the desired amount of n-alkane, such electron donor may also be introduced recombinantly. Guidelines for optimizing electron donors for the reaction catalyzed by the recombinant ADM proteins described herein may be summarized as follows:

    
    
        1. In cyanobacteria, electrons are shuttled from photosystem I to ferredoxin and from ferredoxin to the ADM enzyme.
        2. In bacteria that lack photosystem I, electrons can be shuttled from NADPH to ferredoxin via the action of ferredoxin-NADP+ reductase (EC 1.18.1.2) and from ferredoxin to the ADM enzyme.
        3. In bacteria that lack photosystem I, electrons can be shuttled from NADPH to flavodoxin via the action of ferredoxin-NADP+ reductase (EC 1.18.1.2) and from flavodoxin to the ADM enzyme.
        4. In bacteria that lack photosystem I, electrons can be shuttled from NADH to ferredoxin via the action of Trichomonas vaginalis NADH dehydrogenase and from ferredoxin to the ADM enzyme.
        5. In all bacteria, electrons can be shuttled from pyruvate to ferredoxin by the action of pyruvate:ferredoxin oxidoreductase (EC 1.2.7.1), and from ferredoxin to the ADM enzyme.
    
    


In addition to the in vivo production of n-alkanes discussed above, AAR and ADM proteins encoded by the genes listed in Tables 1 and 2 can be purified. When incubated in vitro with an appropriate electron donor (e.g., a ferredoxin, as discussed above), the proteins will catalyze the enzymatic synthesis of n-alkanes in vitro from appropriate starting materials (e.g., an acyl-ACP or n-alkanal).
In addition to the pathways for n-alkane synthesis described above, the invention also provides an alternative pathway, namely, acyl-CoA→n-alkanal→(n−1)-alkane, via the successive activities of acyl-CoA reductase (ACR) and ADM. Normally, acyl-CoA is the first intermediate in metabolic pathways of fatty acid oxidation; thus, upon import into the cell, exogenously added free fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (FIG. 1B). Acyl-CoA can also be derived purely biosynthetically as follows: acyl-ACP→free fatty acid→acyl-CoA, via the activities of cytoplasmic acyl-ACP thioesterase (EC 3.1.2.14; an example is leader-signal-less E. coli TesA) and the endogenous and/or heterologous acyl-CoA synthetase. Thus, in one embodiment, the invention provides a method for the biosynthesis of n-alkanes via the pathway: acyl-ACP→intracellular free fatty acid→acyl-CoA→n-alkanal→(n−1)-alkane (FIG. 1D), catalzyed by the successive activities of acyl-ACP thioesterase, acyl-CoA synthetase, acyl-CoA reductase, and ADM. For example, the acyl-CoA reductase Acr1 from Acinetobacter calcoaceticus and the ADM from Synechococcus sp. PCC7942 (SYNPCC7942—1593) can be used to transform E. coli, which is cultured in the presence of exogenous free fatty acids. The free fatty acids are taken up by the cells as acyl-CoA, which are then converted to n-alkanal by Acr1, and thence to (n−1)-alkane by ADM.
EXAMPLE 2
Production of n-Alkanes, n-Alkenes, and Fatty Alcohols in Escherichia coli K-12 Through Heterologous Expression of Synechococcus elongatus PCC7942 SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)
The natural SYNPCC7942—1593-SYNPCC7942—1594 operonic sequence was PCR-amplified from the genomic DNA of Synechococcus elongatus PCC7942 and cloned into the pAQ1 homologous recombination vector pJB5 via NdeI and EcoRI. The resulting plasmid was denoted pJB823. This construct placed the SYNPCC7942—1593-SYNPCC7942—1594 operon under the transcriptional control of the constitutive aphII promoter. The sequence of pJB823 is provided as SEQ ID NO: 15. The intracellular hydrocarbon products of E. coli K-12 EPI400TM (Epicentre) harboring pJB823, JCC1076, were compared to those of EPI400TM harboring pJB5, the control strain JCC9a, by gas chromatography-mass spectrometry (GC-MS). Clonal cultures of JCC9a and JCC1076 were grown overnight at 37° C. in Luria Broth (LB) containing 2% glucose, 100 μg/ml carbenicillin, 50 μg/ml spectinomycin, 50 μg/ml streptomycin, and 1× CopyCutter Induction Solution (Epicentre). For each strain, 15 ml of saturated culture was collected by centrifugation. Cell pellets were washed thoroughly by three cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by three cycles of microcentrifugation and aspiration. Cell pellets were then extracted by vortexing for five minutes in 0.8 ml acetone containing 100 μg/mlbutylated hydroxytoluene (BHT; a general antioxidant) and 100 μg/ml ethyl arachidate (EA; an internal reporter of extraction efficiency). Cell debris was pelleted by centrifugation, and 700 μl extractant was pipetted into a GC vial. These JCC9a and JCC1067 acetone samples, along with authentic standards, were then analyzed by GC-MS.
The gas chromatograph was an Agilent 7890A GC equipped with a 5975C electron-impact mass spectrometer. Liquid samples (1.0 μl) were injected into the GC with a 7683 automatic liquid sampler equipped with a 10 μl syringe. The GC inlet temperature was 290° C. and split-less injection was used. The capillary column was an Agilent HP-5MS (30 m×0.25 mm×0.25 μm). The carrier gas was helium at a flow rate of 1.0 ml/min. The GC oven temperature program was 50° C., hold 1 min/10° C. per min to 290° C./hold 9 min. The GC-MS interface temperature was 290° C. The MS source temperature was 230° C., and the quadrapole temperature was 150° C. The mass range was 25-600 amu. MS fragmentation spectra were matched against the NIST MS database, 2008 version.
Peaks present in the total-ion GC-MS chromatograms were chemically assigned in one of two ways. In the first, assignment was done by ensuring that both the retention time and the fragmentation mass spectrum corresponded to the retention time and fragmentation mass spectrum, respectively, of an authentic standard—this is referred to as “Method 1”, and is essentially unambiguous. In the absence of authentic standards, only a tentative chemical assignment can be reached; this was done by collectively integrating the following data for the peak in question: (i) the structure of the fragmentation spectrum, especially with regard to the weight of the molecular ion, and to the degree to which it resembled a hydrocarbon-characteristic “envelope” mass spectrum, (ii) the retention time, especially with regard to its qualitative compatibility with the assigned compound, e.g., cis-unsaturated n-alkenes elute slightly before their saturated n-alkane counterparts, and (iii) the likelihood that the assigned compound is chemically compatible with the operation of the AAR-ADM and related pathways in the host organism in question, e.g., fatty aldehydes generated by AAR are expected to be converted to the corresponding fatty alcohols by host dehydrogenases in E. coli if they are not acted upon sufficiently quickly by ADM. This second approach to peak assignment is referred to as “Method 2”. In the total-ion GC-MS chromatogram in FIG. 2, as well as in all such chromatograms in subsequent figures, peaks chemically assigned by Method 1 are labeled in regular font, whereas those assigned by Method 2 are labeled in italic font.
Total ion chromatograms (TICs) of JCC9a and JCC1076 acetone cell pellet extractants are shown in FIG. 2. The TICs of C8-C20 n-alkane authentic standards (Sigma 04070), as well as 1-tetradecanol (Sigma 185388) plus 1-hexadecanol (Sigma 258741) plus 1-octadecanol (Sigma 258768), are also shown. Hydrocarbons identified in JCC1076, but not in control strain JCC9a, are detailed in Table 3. These hydrocarbons are n-pentadecane (1), 1-tetradecanol (1), n-heptadecene (2), n-heptadecane (1), and 1-hexadecanol (1), where the number in parentheses indicates the GC-MS peak assignment method. MS fragmentation spectra of the Method 1 peaks are shown in FIG. 3, plotted against their respective library hits.







TABLE 3







Hydrocarbons detected by GC-MS in acetone cell pellet extractants


of JCC1076 but not JCC9a, in increasing order of retention time.














GC-MS Peak
Candidate


Compound
JCC9a
JCC1076
Assigment
isomer





n-pentadecane
−
+
Method 1



1-tetradecanol
−
+
Method 1


n-heptadecene
−
+
Method 2
cis-7-





(envelope-type MS
heptadecene





with molecular





ion mass 238)


n-pentadecane
−
+
Method 1


1-hexadecanol
−
+
Method 1





“−” not detected;


“+” detected.






The formation of these five products is consistent with both the expected incomplete operation, i.e., acyl-ACP→fatty aldehyde→fatty alcohol, and expected complete operation, i.e., acyl-ACP fatty aldehyde→alkane/alkene, of the AAR-ADM pathway in E. coli, whose major straight-chain acyl-ACPs include 12:0, 14:0, 16:0, 18:0, 16:1Δ9cis, and 18:1Δ11cis acyl groups (Heipieper H J (2005); Appl Environ Microbiol 71:3388). Assuming that n-heptadecene (2) is derived 18:1Δ11 cis-ACP, it would correspond to cis-7-heptadecene. Indeed, an n-heptadecene isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 238; also, as expected, it elutes slightly before n-heptadecane.
EXAMPLE 3
Production of n-Alkanes, n-Alkenes, and Fatty Alcohols in Escherichia coli B Through Heterologous Expression of Synechococcus elongatus PCC7942 SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)
The natural SYNPCC7942—1593-SYNPCC7942—1594 operonic sequence was excised from pJB823 using NdeI and EcoRI, and cloned into the commercial expression vector pCDFDuetTM-1 (Novagen) cut with via NdeI and MfeI. The resulting plasmid was denoted pJB855 (SEQ ID NO: 16). This construct placed the SYNPCC7942—1593-SYNPCC7942—1594 operon under the transcriptional control of the inducible T7lacO promoter.
The intracellular hydrocarbon products of E. coli BL21(DE3) (Novagen) harboring pJB855, JCC1113, were compared to those of E. coli. BL21(DE3) harboring pCDFDuetTM-1, the control strain JCC114, by gas chromatography-mass spectrometry (GC-MS). Starter clonal cultures of JCC1114 and JCC1113 were grown overnight at 37° C. in M9 minimal medium supplemented with 6 mg/l FeSO4.7H2O, 50 μg/ml spectinomycin, and 2% glucose as carbon source; this medium is referred to M9fs. Each starter culture was used to inoculate a 32 ml culture of M9fs at an initial OD600 of 0.1. Inoculated cultures were grown at 37° C. at 300 rpm until an OD600 of 0.4 has been reached, at which point IPTG was added to a final concentration of 1 mM. After addition of inducer, cultures were grown under the same conditions for an additional 17 hours. For each strain, 12 ml of saturated culture was then collected by centrifugation. Cell pellets were washed thoroughly by 3 cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by 3 cycles of microcentrifugation and aspiration. Cell pellets were then extracted by vortexing for 5 minutes in 0.7 ml acetone containing 20 μg/ml BHT and 20 μg/ml EA. Cell debris was pelleted by centrifugation, and 600 μl supernatant was pipetted into a GC vial. These JCC1114 and JCC1113 samples, along with authentic standards, were then analyzed by GC-MS as described in Example 2. The TICs of JCC1114 and JCC1113 acetone cell pellet extractants are shown in FIG. 4; n-alkane and 1-alkanol standards are as in Example 2. Hydrocarbons identified in JCC1113, but not in control strain JCC1114, are detailed in Table 4.







TABLE 4







Hydrocarbons detected by GC-MS in acetone cell pellet extractants of JCC1113 but not


JCC1114 in increasing order of retention time.











Compound
JCC1114
JCC1113
GC-MS Peak Assigment
Candidate isomer





n-tridecane
−
+
Method 1



n-tetradecane
−
+
Method 1


n-pentadecene
−
+
Method 2 (envelope-type MS
cis-7-pentadecene





with molecular ion mass 210)


1-dodecanol
−
+
Method 2


n-pentadecane
−
+
Method 1


n-hexadecene
−
+
Method 2 (envelope-type MS
cis-8-hexadecene





with molecular ion mass 224)


n-hexadecane
−
+
Method 1


1-tetradecanol
−
+
Method 1


n-heptadecene
−
+
Method 2 (envelope-type MS
cis-7-heptadecene





with molecular ion mass 238)


n-heptadecane
−
+
Method 1


1-pentadecanol
−
+
Method 2


1-hexadecenol
−
+
Method 2
cis-9-hexadecen-1-ol


1-hexadecanol
−
+
Method 1


1-octadecenol
−
+
Method 2 (envelope-type MS
cis-11-octadecen-1-ol





with molecular ion mass 250)





“−” not detected;


“+” detected.






These hydrocarbons are n-tridecane (1), n-tetradecane (1), n-pentadecene (2), 1-dodecanol (2), n-pentadecane (1), n-hexadecene (2), n-hexadecane (1), 1-tetradecanol (1), n-heptadecene (2), n-heptadecane (1), 1-pentadecanol (2), 1-hexadecenol (2), 1-hexadecanol (1), and 1-octadecenol (2), where the number in parentheses indicates the GC-MS peak assignment method. MS fragmentation spectra of Method 1 peaks are shown in FIG. 5, plotted against their respective library hits. The major products were n-pentadecane and n-heptadecene.
The formation of these fourteen products is consistent with both the expected incomplete operation, i.e., acyl-ACP→fatty aldehyde→fatty alcohol, and expected complete operation, i.e., acyl-ACP→fatty aldehyde→alkane/alkene, of the Aar-Adm pathway in E. coli, whose major straight-chain acyl-ACPs include 12:0, 14:0, 16:0, 18:0, 16:1Δ9cis, and 18:1Δ11cis acyl groups (Heipieper H J (2005). Adaptation of Escherichia coli to Ethanol on the Level of Membrane Fatty Acid Composition. Appl Environ Microbiol 71:3388). Assuming that n-pentadecene (2) is derived 16:1Δ9cis-ACP, it would correspond to cis-7-pentadecene. Indeed, an n-pentadecene isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 210; also, as expected, it elutes slightly before n-pentadecane. With respect to 1-dodecanol (2), a sufficiently clean fragmentation spectrum could not be obtained for that peak due to the overlapping, much larger n-pentadecane (1) peak. Its presence, however, is consistent with the existence of 12:0-ACP in E. coli, and its retention time is exactly that extrapolated from the relationship between 1-alkanol carbon number and observed retention time, for the 1-tetradecanol, 1-hexadecanol, and 1-octadecanol authentic standards that were run. Assuming that n-hexadecene (2) is derived from the trace-level unsaturated 17:1Δ9cis acyl group expected in the E. coli acyl-ACP population due to rare acyl chain initiation with propionyl-CoA as opposed to malonyl-CoA, it would correspond to cis-8-hexadecene. Indeed, an n-hexadecene isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 224; also, as expected, it elutes slightly before n-hexadecane. Assuming that n-heptadecene (2) is derived 18:1Δ11cis-ACP, it would correspond to cis-7-heptadecene. Indeed, an n-heptadecene isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 238; also, as expected, it elutes slightly before n-heptadecane. With respect to 1-pentadecanol (2), a sufficiently clean fragmentation spectrum could not be obtained for that peak due to its low abundance. Its presence, however, is consistent with the existence of trace-level 15:0 acyl group expected in the E. coli acyl-ACP population due to rare acyl chain initiation with propionyl-CoA as opposed to malonyl-CoA, and its retention time is exactly that interpolated from the relationship between 1-alkanol carbon number and observed retention time, for the 1-tetradecanol, 1-hexadecanol, and 1-octadecanol authentic standards that were run. In addition, 1-pentadecanol was identified as the highest-confidence MS fragmentation library hit at that retention time in acetone extracts of JCC1170, a BL21(DE3) derivative that expresses Aar without Adm (see Example 4). With respect to 1-hexadecenol (2), a sufficiently clean fragmentation spectrum could not be obtained for that peak due to its low abundance; however, assuming that it is derived 16:1Δ9cis-ACP, it would correspond to cis-9-hexadecen-1-ol. Also, as expected, it elutes slightly before 1-hexadecanol. Finally, assuming that n-octadecenol (2) is derived 18:11Δ9cis-ACP, it would correspond to cis-11-octadecen-1-ol. Indeed, an n-octadecen-1-ol isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 250; also, as expected, it elutes slightly before 1-octadecanol.
EXAMPLE 4
Production of Fatty Alcohols in Escherichia coli B Through Heterologous Expression of Synechococcus elongatus SYNPCC7942—1594 (aar) without Co-Expression of SYNPCC7942—1593 (adm)
In order to test the hypothesis that both AAR and ADM are required for alkane biosynthesis, as well as the prediction that expression of AAR alone should result in the production of fatty alcohols only in E. coli (due to non-specific dehydrogenation of the fatty aldehydes generated), expression constructs containing just SYNPCC7942—1593 (ADM) and just SYNPCC7942—1594 (AAR), were created. Accordingly, the SYNPCC7942—1593 and SYNPCC7942—1594 coding sequences were individually PCR-amplified and cloned via NdeI and MfeI into the commercial expression vector pCDFDuetTM-1 (Novagen). The resulting plasmids were denoted pJB881 (SYNPCC7942—1593 only) and pJB882 (SYNPCC7942—1594 only); in each construct, the coding sequence was placed under the transcriptional control of the inducible T7lacO promoter.
The intracellular hydrocarbon products of E. coli BL21(DE3) (Novagen) harboring pJB881, JCC1169, and of E. coli BL21(DE3) (Novagen) harboring pJB882, JCC1170, were compared to those of E. coli BL21(DE3) harboring pCDFDuetTM-1, the negative control strain JCC114, as well as to the positive control SYNPCC7942—1593-SYNPCC7942—1594 strain JCC1113 (Example 3), by gas chromatography-mass spectrometry (GC-MS). Clonal cultures of JCC1169, JCC1170, JCC1114, and JCC1113 were grown, extracted, and analyzed by GC-MS as described in Example 3, with the following exception: the JCC1170 culture was grown overnight in M9fs medium without IPTG, because the culture did not grow if IPTG was added. Presumably, this was due to the toxic over-accumulation of fatty alcohols that occurred even in the absence of inducer.
The TICs of JCC1169, JCC1170, JCC1114, and JCC1113 acetone cell pellet extractants are shown in FIG. 6; n-alkane and 1-alkanol standard traces have been omitted. Hydrocarbons identified in JCC1170, but not in control strain JCC1114, are detailed in Table 5.







TABLE 5







Hydrocarbons detected by GC-MS in acetone cell pellet extractants of JCC1170 but not


JCC1114 in increasing order of retention time.











Compound
JCC1114
JCC1170
GC-MS Peak Assigment
Candidate isomer





1-tetradecanol
−
+
Method 1



1-pentadecanol
−
+
Method 2 (envelope-type MS





with molecular ion mass 182)


1-hexadecenol
−
+
Method 2 (envelope-type MS
cis-9-hexadecen-1-ol





with molecular ion mass 222)


1-hexadecanol
−
+
Method 1


1-octadecenol
−
+
Method 2 (envelope-type MS
cis-11-octadecen-1-ol





with molecular ion mass 250)





“−” not detected;


“+” detected.






These hydrocarbons are 1-tetradecanol (1), 1-pentadecanol (2), 1-hexadecenol (2), 1-hexadecanol (1), and 1-ocadecenol (2), where the number in parentheses indicates the GC-MS peak assignment method. MS fragmentation spectra of Method 1 peaks are shown in FIG. 7, plotted against their respective library hits. No hydrocarbons were identified in JCC1169, whose trace was indistinguishable from that of JCC1114, as expected owing to absence of fatty aldehyde substrate generation by AAR.
The lack of production of alkanes, alkenes, and fatty alkanols in JCC1169, the production of only fatty alcohols in JCC1170, and the production of alkanes, alkenes, and fatty alkanols in JCC1113 (as discussed in Example 3) are all consistent with the proposed mechanism of alkane biosynthesis by AAR and ADM in E. coli. Thus, the formation of the five fatty alcohols in JCC1170 is consistent with only AAR being active, and active on the known straight-chain acyl-ACPs (see Example 3). With respect to 1-pentadecanol (2), its presence is consistent with the existence of trace-level 15:0 acyl group expected in the E. coli acyl-ACP population due to rare acyl chain initiation with propionyl-CoA as opposed to malonyl-CoA and its retention time is exactly that interpolated from the relationship between 1-alkanol carbon number and observed retention time, for the 1-tetradecanol, 1-hexadecanol, and 1-octadecanol authentic standards that were run. Most importantly, the 1-pentadecanol (2) peak exhibits an envelope-type fragmentation mass spectrum, with the expected molecular ion of molecular weight 182. Unlike in the case of JCC1113, a clean fragmentation spectrum from the candidate 1-hexadecenol peak could now be obtained due to increased abundance. The top library hit was a 1-hexadecenol with the expected molecular ion of molecular weight 222. Assuming that it is derived from 16:1Δ9cis hexadecenyl-ACP, the isomeric assignment would be cis-9-hexadecen-1-ol; also, as expected, it elutes slightly before 1-hexadecanol. Assuming that n-octadecenol (2) is derived 18:11Δ9cis-ACP, it would correspond to cis-11-octadecen-1-ol. Indeed, an n-octadecen-1-ol isomer was identified as the highest-confidence MS fragmentation library hit at that retention time, with the expected molecular ion of molecular weight 250; also, as expected, it elutes slightly before 1-octadecanol. There is also an unidentified side peak in JCC1170 that elutes in the tail of 1-hexadecenol and whose fragmentation mass spectrum was not sufficiently clean to enable possible identification. It is hypothesized that this could be the primary C18 aldehyde product expected of AAR-only activity in E. coli, i.e., cis-11-octadecenal.
EXAMPLE 5
Production of n-Alkanes, n-Alkenes, and Fatty Alcohol in Synechococcus sp. PCC 7002 Through Heterologous Expression of Synechococcus elongatus PCC7942 SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)
In order to test whether heterologous expression of AAR and ADM would lead to the desired alkane biosynthesis in a cyanobacterial host, the SYNPCC7942—1593-SYNPCC7942—1594 operon was expressed in Synechococcus sp. PCC 7002 (JCC138). Accordingly, plasmid pJB823 was transformed into JCC138, generating strain JCC1160. The sequence and annotation of this plasmid is provided as SEQ ID NO: 15, and described in Example 2. In this construct, the SYNPCC7942—1593-SYNPCC7942—1594 operon is placed under the transcriptional control of the constitutive aphII promoter. 500 base pair upstream and downstream homology regions direct homologous recombinational integration into the native high-copy pAQ1 plasmid of JCC138, and an aadA gene permits selection of transformants by virtue of their resistance to spectinomycin.
To test the effect of potentially stronger promoters, constructs directly analogous to pJB823 were also generated that substituted the aphII promoter with the following: the promoter of cro from lambda phage (PcI), the promoter of cpcB from Synechocystis sp. PCC 6803 (PcpcB), the trc promoter along with an upstream copy of a promoter-lacI cassette (PlacI-trc), the synthetic EM7 promoter (PEM7). Promoters were exchanged via the NotI and NdeI sites flanking the promoter upstream of the SYNPCC7942—1593-SYNPCC7942—1594 operon in the pJB823 vector. The corresponding final plasmids were as follows: pJB886 (Pcl), pJB887 (PcpcB), pJB889 (PlacI-trc), pJB888 (PEM7), and pJB823 (PaphII). These sequences of pJB886, pJB887, pJB889, and pJB888 are identical to the sequence of pJB823 except in the region between the NotI and NdeI sites, where they differ according to the promoter used. The sequences of the different promoter regions are provided as SEQ ID NO: 19 (PcI), SEQ ID NO: 20 (PcpcB), SEQ ID NO: 21 (PlacI-trc), and SEQ ID NO: 22 (PEM7). The sequence of the PaphII promoter is presented in SEQ ID NO: 15.
pJB886, pJB887, pJB889, pJB888, pJB823, as well as pJB5 (the empty pAQ1 targeting vector that entirely lacked the SYNPCC7942—1593-SYNPCC7942—1594 operonic sequence) were naturally transformed into JCC138 using a standard cyanobacterial transformation protocol, generating strains JCC1221 (PcI), JCC1220 (PcpcB), JCC1160b (PlacI-trc), JCC1160a (PEM7), JCC1160 (PaphII), and JCC879 (pJB5), respectively. Briefly, 5-10 μg of plasmid DNA was added to 1 ml of neat JCC138 culture that had been grown to an OD730 of approximately 1.0. The cell-DNA mixture was incubated at 37° C. for 4 hours in the dark with gentle mixing, plated onto A+ plates, and incubated in a photoincubator (Percival) for 24 hours, at which point spectinomycin was underlaid to a final concentration of 50 μg/ml. Spectinomycin-resistant colonies appeared after 5-8 days of further incubation under 24 hr-light conditions ( ̃100 μmol photons m−2 s−1). Following one round of colony purification on A+ plates supplemented with 100 μg/ml spectinomycin, single colonies of each of the six transformed strains were grown in test-tubes for 4-8 days at 37° C. at 150 rpm in 3% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator. The growth medium used for liquid culture was A+ with 200 μg/ml spectinomycin.
In order to compare the intracellular hydrocarbon products of strains JCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160, and JCC879, 24 OD730-ml worth of cells ( ̃2.4×109 cells) of each strain was collected from the aforementioned test-tube cultures by centrifugation. Cell pellets were washed thoroughly by 3 cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by 3 cycles of microcentrifugation and aspiration. Cell pellets were then extracted by vortexing for 5 minutes in 0.7 ml acetone containing 20 μg/ml BHT and 20 μg/ml EA. Cell debris was pelleted by centrifugation, and 600 supernatant was pipetted into a GC vial. The six extractants, along with authentic standards, were then analyzed by GC-MS as described in Example 2.
The TICs of JCC1221, JCC1220, JCC1160b, JCC1160a, JCC1160, and JCC879 acetone cell pellet extractants are shown in FIG. 8; n-alkane and 1-alkanol standards are as in Example 2. Consistent with a range of promoter strengths, and with function of the AAR-ADM pathway, there was a range of hydrocarbon accumulation, the order of accumulation being PcI>PcpcB>PlacI-trc>PEM7>PaphII (FIG. 8A).
In JCC1160, approximately 0.2% of dry cell weight was found as n-alkanes and n-alkan-1-ol (excluding n-nonadec-1-ene). Of this 0.2%, approximately three-quarters corresponded to n-alkanes, primary products being n-heptadecane and n-pentadecane. These hydrocarbons were not detected in JCC879. The data are summarized in Table 6A.







TABLE 6A







Hydrocarbons detected in acetone extracts of JCC1160 and JCC879.










Approximate % of




dry cell weight











Compound
JCC879
JCC1160







n-pentadecane
not detected
0.024%



n-hexadecane
nd
0.004%



n-heptadecane
nd
0.110%



n-octadecan-1-ol
nd
0.043%










Total
0.181%



% of products that are n-alkanes
  76%










The highest accumulator was JCC1221 (PcI). Hydrocarbons identified in JCC1221, but not in control strain JCC879, are detailed in Table 6B, Table 6C and FIG. 8B. These hydrocarbons are n-tridecane (1), n-tetradecane (1), n-pentadecene (2), n-pentadecane (1), n-hexadecane (1), n-heptadec-di-ene (2), three isomers of n-heptadecene (2), n-heptadecane (1), and 1-ocadecanol (1), where the number in parentheses indicates the GC-MS peak assignment method.







TABLE 6B







n-Alkanes quantitated in acetone extract of JCC1221










Compound
% of JCC1221 dry cell weight







n-tridecane
<0.001% 



n-tetradecane
0.0064% 



n-pentadecane
0.40%



n-hexadecane
0.040% 



n-heptadecane
 1.2%



Total
1.67%










MS fragmentation spectra of Method 1 peaks are shown in FIG. 9, plotted against their respective library hits. The only alkanes/alkenes observed in JCC879 were 1-nonadecene and a smaller amount of nonadec-di-ene, alkenes that are known to be naturally synthesized by JCC138 (Winters K et al. (1969) Science 163:467-468). The major products observed in JCC1221 were n-pentadecane ( ̃25%) and n-heptadecane ( ̃75%); all others were in relatively trace levels.
The formation of n-pentadecane and n-heptadecane in JCC1221, as well as the nine other trace hydrocarbon products, is consistent with the virtually complete operation of the ADM-AAR pathway in JCC138, i.e., 16:0 hexadecyl-ACP→n-hexadecanal→n-pentadecane and 18:0 octadecyl-ACP→n-octadecanal→n-heptadecane. Indeed it is known that the major acyl-ACP species in this organism are C16:0 and C18:0 (Murata N et al. (1992) Plant Cell Physiol 33:933-941). Relatively much less fatty alcohol is produced relative to AAR-ADM expression in E. coli (Example 3), as expected given the presence in JCC138 of a cyanobacterial ferredoxin/ferredoxin-NADPH reductase system that can regenerate the di-iron active site of ADM, thereby preventing the accumulation of hexadecanal and octadecanal that could in turn be non-specifically dehydrogenated to the corresponding 1-alkanols. Thus, in JCC1221, only a very small 1-octadecanol (1) peak is observed (FIG. 8).
The other trace hydrocarbons seen in JCC1221 are believed to be unsaturated isomers of n-pentadecane and n-heptadecane (Table 6C). It is hypothesized that all these alkenes are generated by desaturation events following the production of the corresponding alkanes by the SYNPCC7942—1593 Adm. This contrasts with the situation in E. coli, where double bonds are introduced into the growing acyl chain while it is linked to the acyl carrier protein (Example 3). JCC138 is known to have a variety of position-specific acyl-lipid desaturases that, while nominally active only on fatty acids esterified to glycerolipids, could potentially act on otherwise unreactive alkanes produced nonphysiologically by the action of AAR and ADM. JCC138 desaturases, i.e., DesA, DesB, and DesC, introduce cis double bonds at the Δ9, Δ12, and Δ15 positions of C18 acyl chains, and at the Δ9 and Δ12 positions of C16 acyl chains (Murata N and Wada H (1995) Biochem J. 308:1-8). The candidate n-pentadecene peak is believed to be cis-4-pentadecene (Table 6C).
Assuming also that heptadecane could also serve as a substrate for JCC138 desaturases, and that it would be desaturated at positions analogous to the Δ9, Δ12, and Δ15 of the C18 acyl moiety, there are four theoretically possible mono-unsaturated isomers: cis-3-heptadecene, cis-6-heptadecene, cis-8-heptadecene, and cis-9-heptadecene. These isomers do not include the single n-heptadecene species nominally observed in E. coli, cis-7-heptadecene (Example 2). It is believed that the three peaks closest to the n-heptadecane peak—denoted by subscripts 1, 2, and 3 in Table 6C and FIG. 8B-encompass at least three of these four mono-unsaturated heptadecane isomers. Consistent with this, n-heptadecene2 and n-heptadecene3 peaks have the expected molecular ions of mass 238 in their envelope-type fragmentation spectra. There are many isomeric possibilities, accordingly, for the putative cis,cis-heptadec-di-ene peak, which has an envelope-type fragmentation spectrum with the expected molecular ions of mass 236. As expected, all putative heptadecene species elute slightly before n-heptadecane.







TABLE 6C







Alkane and alkenes detected by GC-MS in acetone cell pellet extractants


of JCC1221 but not JCC879 in increasing order of retention time.














GC-MS Peak



Compound
JCC879
JCC1221
Assigment
Candidate isomer





n-tridecane
−
+
Method 1



n-tetradecane
−
+
Method 1


n-pentadecene
−
+
Method 2
cis-4-pentadecene


n-pentadecane
−
+
Method 1


n-hexadecane
−
+
Method 1


n-heptadec-di-ene
−
+
Method 2 (envelope-
cis,cis-heptadec-di-ene





type MS with





molecular ion mass





236)


n-heptadecene3
−
+
Method 2 (envelope-
cis-[3/6/8/9]-





type MS with
heptadecene





molecular ion mass





238)


n-heptadecene2
−
+
Method 2 (envelope-
cis-[3/6/8/9]-





type MS with
heptadecene





molecular ion mass





238)


n-heptadecene1
−
+
Method 2
cis-[3/6/8/9]-






heptadecene


n-heptadecane
−
+
Method 1


1-octadecanol
−
+
Method 1





“−”, not detected;


“+”, detected.






EXAMPLE 6
Intracellular Accumulation of n-Alkanes to up to 5% of Dry Cell Weight in Synechococcus sp. PCC 7002 Through Heterologous Expression of Synechococcus elongatus PCC7942 SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)
In order to quantitate more accurately the level of intracellular accumulation of n-alkane products in the alkanogen JCC1221 (Example 5), the levels of n-pentadecane and n-heptadecane, as well as the relatively trace products n-tetradecane and n-hexadecane, were quantified with respect to dry cell weight (DCW). Based on the hypothesis that the extent of n-alkane production could correlate positively with the level of SYNPCC7942—1593-SYNPCC7942—1594 operon expression, the DCW-normalized n-alkane levels of JCC1221 were determined as a function of the spectinomycin concentration of the growth medium. The rationale was that the higher the spectinomycin selective pressure, the higher the relative copy number of pAQ1, and the more copies of the aadA-linked SYNPCC7942—1593-SYNPCC7942—1594 operon.
A clonal starter culture of JCC1221 was grown up in A+ medium supplemented with 100 μg/ml spectinomycin in for 7 days at 37° C. at 150 rpm in 3% CO2-enriched air at 100 mmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator. At this point, this culture was used to inoculate triplicate 30 ml JB2.1 medium (PCT/US2009/006516) flask cultures supplemented with 100, 200, 300, 400, or 600 μg/ml spectinomycin. JB2.1 medium consists of 18.0 g/l sodium chloride, 5.0 g/1 magnesium sulfate heptahydrate, 4.0 g/l sodium nitrate, 1.0 g/l Tris, 0.6 g/l potassium chloride, 0.3 g/l calcium chloride (anhydrous), 0.2 g/l potassium phosphate monobasic, 34.3 mg/l boric acid, 29.4 mg/l EDTA (disodium salt dihydrate), 14.1 mg/l iron (III) citrate hydrate, 4.3 mg/l manganese chloride tetrahydrate, 315.0 μg/l zinc chloride, 30.0 μg/1 molybdenum (VI) oxide, 12.2 μg/l cobalt (II) chloride hexahydrate, 10.0 μg/l vitamin B12, and 3.0 μg/1 copper (II) sulfate pentahydrate. The 15 cultures were grown for 10 days at 37° C. at 150 rpm in 3% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator.
For each culture, 5-10 ml was used for dry cell weight determination. To do so, a defined volume of culture—corresponding to approximately 20 mg DCW—was centrifuged to pellet the cells. Cells were transferred to a pre-weighed eppendorf tube, and then washed by 2 cycles of resuspension in Milli-Q water and microcentrifugation, and dewetted by 3 cycles of microcentrifugation and aspiration. Wet cell pellets were frozen at −80° C. for two hours and then lyophilized overnight, at which point the tube containing the dry cell mass was weighed again such that the mass of the cell pellet could be calculated within ±0.1 mg. In addition, for each culture, 0.3-0.8 ml was used for acetone extraction of the cell pellet for GC analysis. To do so, a defined volume of culture—corresponding to approximately 1.4 mg DCW—was microcentrifuged to pellet the cells. Cells were then washed by 2 cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted by 4 cycles of microcentrifugation and aspiration. Dewetted cell pellets were then extracted by vortexing for 1 minute in 1.0 ml acetone containing 50 μg/ml BHT and 160 μg/mln-heptacosane internal standard (Sigma 51559). Cell debris was pelleted by centrifugation, and 700 μl supernatant was pipetted into a GC vial.
Concentrations of n-tetradecane, n-pentadecane, n-hexadecane, and n-heptadecane in the fifteen extractants were quantitated by gas chromatography/flame ionization detection (GC/FID). Unknown n-alkane peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known n-tetradecane, n-pentadecane, n-hexadecane, and n-heptadecane authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes. n-Alkane production was calculated as a percentage of the DCW extracted in acetone.
Consistent with scaling between pAQ1 selective pressure and the extent of intracellular n-alkane production in JCC1221, there was a roughly positive relationship between the % n-alkanes with respect to DCW and spectinomycin concentration (FIG. 10). For all JCC1221 cultures, n-alkanes were  ̃25% n-pentadecane and  ̃75% n-heptadecane. The minimum n-alkane production was  ̃1.8% of DCW at 100 μg/ml spectinomycin and 5.0% in one of the 600 μg/ml spectinomycin cultures.
EXAMPLE 7
Production of n-Alkanes in Synechococcus sp. PCC 7002 Through Heterologous Expression of Prochlorococcus marinus MIT 9312 PMT9312—0532 (adm) and PMT9312—0533 (aar)
This candidate Adm/Aar pair from Prochlorococcus marinus MIT9312 was selected for functional testing by heterologous expression in JCC138 because of the relatively low amino acid homology (62%) of these proteins to their Synechococcus elongatus PCC7942 counterparts, SYNPCC7942—1593 and SYNPCC7942—1594. Specifically, the 252-amino acid protein PMT9312—0532 exhibits only 62% amino acid identity with the 232 amino acid protein SYNPCC7942—1593, wherein amino acids 33-246 of the former are aligned with amino acids 11-224 of the latter. The 347 amino acid protein PMT9312—0533 exhibits only 61% amino acid identity with the 342 amino acid protein SYNPCC7942—1594, wherein amino acids 1-337 of the former are aligned with amino acids 1-339 of the latter.
A codon- and restriction-site-optimized version of the PMT9312—0532-PMT9312—0533 operon was synthesized by DNA2.0 (Menlo Park, Calif.), flanked by NdeI and EcoRI sites. The operon was cloned into the pAQ1 homologous recombination vector pJB5 via NdeI and EcoRI, such that the PMT9312—0532-PMT9312—0533 operon was placed under transcriptional control of the aphII promoter. The sequence of the pJB947 vector is provided as SEQ ID NO: 17.
pJB947 was transformed into JCC138 as described in Example 5, generating strain JCC1281. The hydrocarbon products of this strain were compared to those of the negative control strain JCC879, corresponding to JCC138 transformed with empty pJB5 (see Example 5). Eight OD730-ml worth of cells ( ̃8×108 cells) of each strain was collected by centrifugation, having been grown in A+ medium supplemented with 200 μg/ml spectinomycin as described in Example 5. Cell pellets were washed thoroughly by 3 cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by 3 cycles of microcentrifugation and aspiration. Cell pellets were then extracted by vortexing for 5 minutes in 0.7 ml acetone containing 20 μg/ml BHT and 20 μg/ml EA. Cell debris was pelleted by centrifugation, and 600 μl supernatant was pipetted into a GC vial. Samples were analyzed by GC-MS as described in Example 5.
The TICs of JCC1281 and JCC879 acetone cell pellet extractants are shown in FIG. 11; n-alkane standards are as in Example 6. Hydrocarbons identified in JCC1281, but not in control strain JCC879, were n-pentadecane (1) and n-heptadecane (1), where the number in parentheses indicates the GC-MS peak assignment method. MS fragmentation spectra of Method 1 peaks are shown in FIG. 12, plotted against their respective library hits (as noted in Example 5, the only alkanes/alkenes observed in JCC879 were 1-nonadecene and a smaller amount of nonadec-di-ene, alkenes that are known to be naturally synthesized by JCC138). The amount of n-alkanes produced in JCC1281 is at least 0.1% dry cell weight, and at least 2-two times higher than the amount produced by JCC879. The ratio of n-pentadecane:n-heptadecane ( ̃40%: ̃60%) in JCC1281 was higher than that observed in JCC1221 ( ̃25%: ̃75%), suggesting that the PMT9312—0532 (ADM) and/or the PMT9312—0533 (AAR) exhibit higher activity towards the C16 substrates relative to C18 substrates, compared to SYNPCC7942—1593 (ADM) and/or SYNPCC7942—1594 (AAR).
EXAMPLE 8
Augmentation of Native n-Alkane Production in Thermosynechococcus elongatus BP-1 by Overexpression of the Native tll1313 (adm)-tll1312 (aar) Operon
Genes encoding Thermosynechococcus elongatus BP-1 tll1312 (AAR) and tll1313 (ADM) are incorporated into one or more plasmids (e.g., pJB5 derivatives), comprising promoters of differing strength. The plasmids are used to transform Thermosynechococcus elongatus BP-1. Overexpression of the genes in the transformed cells are measured as will the amount of n-alkanes, particularly heptadecane, produced by the transformed cells, in a manner similar to that described in Example 3. The n-alkanes and other carbon-based products of interest can also be isolated from the cell or cell culture, as needed.
Wild-type Thermosynechococcus elongatus BP-1, referred to as JCC3, naturally produces n-heptadecane as the major intracellular hydrocarbon product, with traces of n-hexadecane and n-pentadecane. These n-alkanes were identified by GC-MS using Method 1; fragmentation spectra are shown in FIG. 13. Briefly, a colony of JCC3 was grown in B-HEPES medium to a final OD730 of  ̃4, at which point 5 OD730-ml worth of cells was harvested, extracted in acetone, and analyzed by GC-MS as detailed in Example 5.
In an effort to augment this n-alkane production, the native tll1313-tll1312 operonic sequence from this organism was PCR-amplified and cloned into the Thermosynechococcus elongatus BP-1 chromosomal integration vector pJB825. This construct places the tll1313-tll1312 operon under the transcriptional control of the constitutive cI promoter. The sequence of the resulting plasmid, pJB825t, is shown in SEQ ID NO:18.
pJB825 and pJB825t were naturally transformed into JCC3 using a standard cyanobacterial transformation protocol, generating strains JCC1084 and JCC1084t, respectively. Briefly, 25 μg of plasmid DNA was added to 0.5 ml of concentrated JCC3 culture (OD730  ̃100) that had originally been grown to an OD730 of approximately 1.0 in B-HEPES at 45° C. in 3% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator. The cell-DNA mixture was incubated at 37° C. for 4 hours in the dark with gentle mixing, made up to 7 ml with fresh B-HEPES medium, and then incubated under continuous light conditions ( ̃100 μmol photons m−2 s−1) for 20 hours at 45° C. at 150 rpm in 3% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator. At this point, cells were collected by centrifugation and serial dilutions were mixed with molten top agar and plated on the surface of B-HEPES plates supplemented with 60 μg/ml kanamycin. Transformant colonies appeared in the top agar layer within around 7 days upon incubation in a photoincubator (Percival) in 1% CO2-enriched air at continuous  ̃100 μmol photons m−2 s−1 irradiance. Single colonies of JCC1084 and JCC1084t were then grown up in triplicate to an OD730 of  ̃6 in B-HEPES/60 μg/ml kanamycin liquid culture, and their intracellular hydrocarbon products quantitated by GC-FID.
3.5 OD730-ml worth of cells ( ̃3.5×108 cells) of each replicate culture of each strain was collected by centrifugation. Cell pellets were washed thoroughly by 3 cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by 3 cycles of microcentrifugation and aspiration. Cell pellets were then extracted by vortexing for 1 minutes in 0.7 ml acetone containing 20 μg/ml BHT and 20 μg/ml n-heptacosane. Cell debris was pelleted by centrifugation, and 600 μl supernatant was pipetted into a GC vial. The two extractants, along with authentic C8-C20 n-alkane authentic standards (Sigma 04070), were then analyzed by GC coupled with flame ionization detection (FID) as described in Example 6. Quantitation of n-pentadecane, n-hexadecane, and n-heptadecane by GC-FID, and dry cell weights were taken as described in Example 6.
Consistent with increased expression of tll1313-tll1312 in JCC1084t relative to the control strain JCC1084, n-pentadecane, n-hexadecane, and n-heptadecane were  ̃500%,  ̃100%, and  ̃100% higher, respectively, in JCC1084t relative to their % DCW levels in JCC1084 (FIG. 14). The total n-alkane concentration in both strains was less than 1%. The n-alkane concentration in JCC1084t was at least 0.62% and at least twice as much n-alkane was produced relative to JCC1084.
EXAMPLE 9
Comparison of Intracellular Hydrocarbon Products of JCC1113 (a Derivative of E. coli) and JCC1221 (a Derivative of Synechococcus sp. PCC 7002), Both Strains Heterologously Expressing Synechococcus elongatus SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)
GC-MS TICs of JCC1113 and JCC1221 acetone cell pellet extractants are shown in FIG. 15, along with the TIC of C8-C20 n-alkane authentic standards (Sigma 04070). These two strains are derived from E. coli BL21(DE3) and Synechococcus sp. PCC7002, respectively, and are described in detail in Examples 3 and 5, respectively. JCC1113 synthesizes predominantly n-heptadecene and n-pentadecane, whereas JCC1221 synthesizes predominantly n-heptadecane and n-pentadecane. This figure visually emphasizes the different retention times of the n-heptadecene isomer produced in JCC1113 and n-heptadecane produced in JCC1221.
EXAMPLE 10
Production of Hydrocarbons in Yeast
The methods of the invention can be performed in a number of lower eukaryotes such as Saccharomyces cerevisiae, Trichoderma reesei, Aspergillus nidulans and Pichia pastoris. Engineering such organisms may include optimization of genes for efficient transcription and/or translation of the encoded protein. For instance, because the ADM and AAR genes introduced into a fungal host are of cyanobacterial origin, it may be necessary to optimize the base pair composition. This includes codon optimization to ensure that the cellular pools of tRNA are sufficient. The foreign genes (ORFs) may contain motifs detrimental to complete transcription/translation in the fungal host and, thus, may require substitution to more amenable sequences. The expression of each introduced protein can be followed both at the transcriptional and translational stages by well known Northern and Western blotting techniques, respectively.
Use of various yeast expression vectors including genes encoding activities which promote the ADM or AAR pathways, a promoter, a terminator, a selectable marker and targeting flanking regions. Such promoters, terminators, selectable markers and flanking regions are readily available in the art. In a preferred embodiment, the promoter in each case is selected to provide optimal expression of the protein encoded by that particular ORF to allow sufficient catalysis of the desired enzymatic reaction. This step requires choosing a promoter that is either constitutive or inducible, and provides regulated levels of transcription. In another embodiment, the terminator selected enables sufficient termination of transcription. In yet another embodiment, the selectable/counterselectable markers used are unique to each ORF to enable the subsequent selection of a fungal strain that contains a specific combination of the ORFs to be introduced. In a further embodiment, the locus to which relevant plasmid construct (encoding promoter, ORF and terminator) is localized, is determined by the choice of flanking region.
The engineered strains can be transformed with a range of different genes for production of carbon-based products of interest, and these genes are stably integrated to ensure that the desired activity is maintained throughout the fermentation process. Various combinations of enzyme activities can be engineered into the fungal host such as the ADM, ADR pathways while undesired pathways are attenuated or knocked out.
EXAMPLE 11
Quantitation of Intracellular n-pentadecane:n-heptadecane Ratio of Synechococcus sp. PCC 7002 Strains Constitutively Expressing Heterologous Synechococcus elongatus SYNPCC7942—1593 (adm) plus SYNPCC7942—1594 (aar) or Heterologous Prochlorococcus marinus MIT 9312 PMT9312—0532 (adm) Plus PMT9312—0533 (aar) on pAQ1
In Example 5 (“Production of n-Alkanes, n-Alkenes, and Fatty Alcohol in Synechococcus sp. PCC 7002 through Heterologous Expression of Synechococcus elongatus PCC7942 SYNPCC7942—1593 (adm) and SYNPCC7942—1594 (aar)”) and Example 7 (“Production of n-Alkanes in Synechococcus sp. PCC 7002 through Heterologous Expression of Prochlorococcus marinus MIT 9312 PMT9312—0532 (adm) and PMT9312—0533 (aar)”), the intracellular hydrocarbon products of JCC138 (Synechococcus sp. PCC 7002) strains expressing the Synechococcus elongatus sp. PCC7942 and Prochlorococcus marinus MIT 9312 adm-aar operons were analyzed by GC-MS. In this Example, GC-FID (Gas Chromatography-Flame Ionization Detection) was applied to more accurately measure these products with respect to dry cell weight. Of special interest was the ratio between n-pentadecane and n-heptadecane. In this regard, it is noted that Synechococcus elongatus sp. PCC7942 naturally synthesizes n-heptadecane as the major intracellular n-alkane, whereas Prochlorococcus marinus MIT 9312 naturally synthesizes n-pentadecane as the major intracellular n-alkane.
The following four strains were compared: (1) JCC138, corresponding to wild-type Synechococcus sp. PCC 7002, (2) JCC879, corresponding to negative control strain JCC138 transformed with pAQ1-targeting plasmid pJB5 described in Example 5, (3) JCC1469, corresponding to JCC138 ΔSYNPCC7002_A1173::gent (JCC1218) transformed with pAQ1-targeting plasmid pJB886 encoding constitutively expressed Synechococcus elongatus sp. PCC7942 adm-aar described in Example 5, and (4) JCC1281, corresponding to JCC138 transformed with pAQ1-targeting plasmid pJB947 encoding constitutively expressed Prochlorococcus marinus MIT 9312 adm-aar, described in Example 7. A clonal starter culture of each strain was grown up for 5 days at 37° C. at 150 rpm in 2% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator in A+ (JCC138), A+ supplemented with 100 μg/ml spectinomycin (JCC879 and JCC1281), or A+ supplemented with 100 μg/ml spectinomycin and 50 μg/ml gentamycin (JCC1469). At this point, each starter culture was used to inoculate duplicate 30 ml JB2.1 medium flask cultures supplemented with no antibiotics (JCC138) or 400 μg/ml spectinomycin (JCC879, JCC1469, and JCC1281). The eight cultures were then grown for 14 days at 37° C. at 150 rpm in 2% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator.
For each culture, 25 OD730-ml worth of cells was collected by centrifugation in a pre-weighed eppendorf tube. Cells were washed by two cycles of resuspension in Milli-Q water and microcentrifugation, and dewetted by two cycles of microcentrifugation and aspiration. Wet cell pellets were frozen at −80° C. for two hours and then lyophilized overnight, at which point the tube containing the dry cell mass was weighed again such that the mass of the cell pellet ( ̃6 mg) could be calculated within ±0.1 mg. In parallel, 4 OD730-ml worth of cells from each culture was collected by centrifugation in an eppendorf tube, washed thoroughly by three cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by threes cycles of microcentrifugation and aspiration. Dewetted cell pellets were then extracted by vortexing for 15 seconds in 1 ml acetone containing 23.6 mg/l BHT and 24.4 mg/l n-heptacosane (C27) internal standard (ABH); cell debris was pelleted by centrifugation, and 450 μl supernatant was submitted for GC-FID. Acetone-extracted DCW was calculated as 4/25, or 16%, of the DCW measured for 25 OD730-ml worth of cells. In parallel with the eight biological sample extractions, six empty eppendorf tubes were extracted with ABH in the same fashion. The extraction/injection efficiency of all ABH extractants was assessed by calculating the ratio between the n-heptacosane GC-FID peak area of the sample and the average n-heptacosane GC-FID peak area of the six empty-tube controls—only ratios of 100%±3% were accepted (Table 7).
Concentrations of n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), and n-octadecane (C18), in the eight extractants were quantitated by (GC/FID). Unknown n-alkane peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane, and n-octadecane authentic standard concentrations and their corresponding GC-FID peak areas. Based on these linear-regression calibration relationships, 95% confidence intervals (95% CI) were calculated for interpolated n-alkane concentrations in the biological samples; interpolation was used in all cases, never extrapolation. 95% confidence intervals were reported as percentages—95% CI % in Table 1—of the interpolated concentration in question. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 8:1, pressure) and an inlet temperature of 290° C. The column was a HP-5MS (Agilent, 20 m×0.18 mm×0.18 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 80° C., hold 0.3 minutes; 17.6° C./min increase to 290° C.; hold 6 minutes. n-Alkane production was expressed as a percentage of the acetone-extracted DCW. The coefficient of variation of the n-heptacosane GC-FID peak area of the six empty-tube controls was 1.0%.
GC-FID data are summarized in Table 7. As expected, control strains JCC138 and JCC879 made no n-alkanes, whereas JCC1469 and JCC1281 made n-alkanes,  ̃98% of which comprised n-pentadecane and n-heptadecane. JCC1469 made significantly more n-alkanes as a percentage of DCW ( ̃1.9%) compared to JCC1281 ( ̃0.7%), likely explaining the relatively low final OD730 of the JCC1469 cultures. For the duplicate JCC121 cultures expressing Synechococcus elongatus sp. PCC7942 adm-aar, the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane was 26.2% and 25.3%, whereas it was 57.4% and 57.2% for the duplicate JCC1221 cultures expressing Prochlorococcus marinus MIT 9312 adm-aar (Table 7). This result quantitatively confirms that these two different adm-aar operons generate different n-alkane product length distributions when expressed in vivo in a cyanobacterial host.







TABLE 7







Table 7 n-Pentadecane and n-heptadecane quantitated by GC-FID in acetone


cell pellet extractants of JCC138, JCC879, JCC1469, and JCC1281.















C27-normalized
C15 as % of
C17 as % of
(C15 + C17)/(C13 + C14 +





extraction/injection
DCW
DCW
C15 + C16 + C17)
C15/(C15 + C17)


Strain
OD730
efficiency
(95% CI %)
(95% CI %)
Mass %
Mass %
















JCC138 #1
12.5
 98%
nd
nd
na
na


JCC138 #2
13.5
 99%
nd
nd
na
na


JCC879 #1
9.8
100%
nd
nd
na
na


JCC879 #2
8.5
101%
nd
nd
na
na


JCC1469 #1
3.1
101%
0.60% (1.1%)
1.69% (0.7%)
97.8%
26.2%


JCC1469 #2
3.2
102%
0.36% (1.0%)
1.05% (1.1%)
98.0%
25.3%


JCC1281 #1
9.7
101%
0.26% (1.2%)
0.19% (0.9%)
97.2%
57.4%


JCC1281 #2
4.8
101%
0.51% (1.9%)
0.38% (1.1%)
97.2%
57.2%





n-Octadecane was not detected in any of the samples; nd: not detected, na: not applicable.






EXAMPLE 12
Quantitation of Intracellular n-pentadecane:n-heptadecane Ratio of Synechococcus sp. PCC 7002 Strains Inducibly Expressing Chromosomally-Integrated Heterologous Prochlorococcus marinus MIT 9312 PMT9312—0532 (adm) Plus PMT9312—0533 (aar) with or without Heterologous Cyanothece sp. ATCC51142 Cce—0778 (adm) Plus Cce—1430 (aar)
In order to confirm that heterologous expression of Aar and Adm from the chromosome would lead to intracellular n-alkane accumulation, the Prochlorococcus marinus MIT9312 adm-aar operon (encoding PMT9312—0532 plus PMT9312—0533) described in Example 7 was chromosomally integrated at the SYNPCC7002_A0358 locus. To do so, a SYNPCC7002_A0358-targeting vector (pJB1279; SEQ ID NO: 23) was constructed containing 750 bp regions of upstream and downstream homology designed to recombinationally replace the SYNPCC7002_A0358 gene with a spectinomycin-resistance cassette downstream of a multiple cloning site (MCS) situated between said regions of homology. Instead of using a constitutive promoter to express the adm-aar operon, an inducible promoter was employed. Specifically, a urea-repressible, nitrate-inducible nirA-type promoter, P(nir07) (SEQ ID NO:24), was inserted into the MCS via NotI and NdeI, generating the base homologous recombination vector pJB1279.
Two operons were cloned downstream of P(nir07) of pJB1279 to generate two experimental constructs, wherein said operons were placed under transcriptional control of P(nir07). The first operon comprised only the aforementioned Prochlorococcus PMT9312—0532-PMT9312—0533 operon, inserted via NdeI and EcoRI, resulting in the final plasmid pJB286alk_p; the sequence of this adm-aar operon was exactly as described in Example 7. The second operon comprised (1) the same Prochlorococcus PMT9312—0532-PMT9312—0533 adm-aar operon, followed by (2) an adm-aar operon derived from Cyanothece sp. ATCC51142 genes cce—0778 (SEQ ID NO: 31) and cce—1430 (SEQ ID NO: 30), respectively, inserted via EcoRI (selecting the correct orientation by screening), resulting in the final plasmid pJB1256. It is to be noted that Cyanothece sp. ATCC51142 naturally synthesizes n-pentadecane as the major intracellular n-alkane. This Cyanothece adm-aar operon (SEQ ID NO: 25) was codon- and restriction-site-optimized prior to synthesis by DNA2.0 (Menlo Park, Calif.). The operon expresses proteins with amino acid sequences identical to those of the AAR and ADM enzymes from Cyanothece sp. ATCC51142 (SEQ ID NOs: 27 and 29, respectively). The complete operon in plasmid pJB 1256, therefore, comprises 4 genes—ADM and AAR from Prochlorococcus PMT9312 and ADM and AAR from Cyanothece sp. ATCC51142—under the control of a single P(nir07) promoter.
pJB1279, pJB286alk_p, and pJB1256 were naturally transformed into JCC138 exactly as described in Example 5, generating spectinomycin-resistant strains JCC1683c, JCC1683, and JCC1685, respectively. As a first test, a clonal starter culture of each of these three strains, as well as of JCC138, was grown up for 5 days at 37° C. at 150 rpm in 2% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron H (Infors) shaking photoincubator in A+ (JCC138) or A+ supplemented with 100 μg/ml spectinomycin (JCC1683c, JCC1683, and JCC1685). At this point, each starter culture was used to inoculate a 30 ml JB2.1 medium plus 3 mM urea flask culture supplemented with no antibiotics (JCC138) or 100 μg/ml spectinomycin (JCC1683c, JCC1683, and JCC1685). The four cultures were then grown for 14 days at 37° C. at 150 rpm in 2% CO2-enriched air at 100 μmmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator.
20 OD730-ml worth of cells was collected by centrifugation in a pre-weighed eppendorf tube. Cells were washed by two cycles of resuspension in Milli-Q water and microcentrifugation, and dewetted by two cycles of microcentrifugation and aspiration. Wet cell pellets were frozen at −80° C. for two hours and then lyophilized overnight, at which point the tube containing the dry cell mass was weighed again such that the mass of the cell pellet ( ̃6 mg) could be calculated within ±0.1 mg. In parallel, 3.5 OD730-ml worth of cells from each culture was collected by centrifugation in an eppendorf tube, washed thoroughly by three cycles of resuspension in Milli-Q water and microcentrifugation, and then dewetted as much as possible by three cycles of microcentrifugation and aspiration. Dewetted cell pellets were then extracted by vortexing for 15 seconds in. 1.0 ml acetone containing 18.2 mg/l BHT and 16.3 mg/l n-heptacosane (C27) internal standard (ABH); cell debris was pelleted by centrifugation, and 500 μl supernatant was submitted for GC-FID. Acetone-extracted DCW was calculated as 3.5/20, or 17.5%, of the DCW measured for 20 OD730-ml worth of cells. In parallel with the four biological sample extractions, eight empty eppendorf tubes were extracted with ABH in the same fashion. The extraction/injection efficiency of all ABH extractants was assessed by calculating the ratio between the n-heptacosane GC-FID peak area of the sample and the average n-heptacosane GC-FID peak area of the six empty-tube controls—only ratios of 100%±11% were accepted (Table 8).
Concentrations of n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), n-hexadecane (C16), n-heptadecane (C17), and n-octadecane (C18), in the four extractants were quantitated by (GC/FID) as described in Example 11. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure) and an inlet temperature of 290° C. The column was a HP-5 (Agilent, 30 m×0.32 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold 1.0 minute; 10° C./min increase to 290° C.; hold 9 minutes. n-Alkane production was calculated as a percentage of the acetone-extracted DCW. The coefficient of variation of the n-heptacosane GC-FID peak area of the eight empty-tube controls was 3.6%.
GC-FID data are summarized in Table 8. As expected, controls strains JCC138 and JCC1683c made no n-alkanes, whereas JCC683 and JCC1685 made n-alkanes,  ̃97% of which comprised n-pentadecane and n-heptadecane. JCC1685 made significantly more n-alkanes as a percentage of DCW ( ̃0.42%) compared to JCC1683 ( ̃0.16%), likely explaining the relatively low final OD730 of the JCC1685 culture. For JCC1683 expressing Prochlorococcus marinus MIT 9312 adm-aar, the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane was 53.2%, in quantitative agreement with that of JCC1281 expressing the same operon on pAQ1 (57.3%; Table 7). In contrast, for JCC1685 which additionally expresses Cyanothece sp. ATCC51142 adm-aar, the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane was 83.7%. This result demonstrates that the in vivo expression of cce—0778 and cce—1430 in a cyanobacterial host biases the n-alkane product length distribution towards n-pentadecane—even more so than does expression of PMT9312—0532 and PMT9312—0533. The total amount of intracellular n-alkane produced by chromosomal integrants JCC1683 and JCC1685 is apparently lower than that of pAQ1-based transformants such as JCC1469, presumably owing to a combination of lower-copy expression (i.e., chromosome versus high-copy pAQ1), and partially repressed transcription—due to the initial presence of urea in the growth medium—of P(nir07) compared to the constitutive promoters P(aphII) (JCC1281) and P(cI) (JCC1469).







TABLE 8







Table 8 n-Pentadecane and n-heptadecane quantitated by GC-FID in acetone


cell pellet extractants of JCC138, JCC1683c, JCC1683, and JCC1685.















C27-normalized
C15 as % of
C17 as % of
(C15 + C17)/(C13 + C14 +





extraction/injection
DCW
DCW
C15 + C16 + C17)
C15/(C15 + C17)


Strain
OD730
efficiency
(95% CI %)
(95% CI %)
Mass %
Mass %





JCC138
17.0
110%
nd
nd
na
na


JCC1683c
13.4
108%
nd
nd
na
na


JCC1683
12.2
111%
0.083% (7.6%) 
0.073% (12.5%)
97.3%
53.2%


JCC1685
10.0
110%
0.341% (13.0%)
0.066% (8.8%) 
96.7%
83.7%





n-Octadecane was not detected in any of the samples; nd: not detected, na: not applicable.






In order to confirm the urea-repressibility/nitrate-inducibility of P(nir07), the intracellular n-alkane product distribution of JCC1685 was determined from cultures grown in either JB2.1 medium, containing only nitrate as the nitrogen source, and JB2.1 supplemented with 6 mM urea, urea being preferentially utilized as nitrogen source relative to nitrate and provided at a concentration such that it became depleted when the culture reached an OD730 of  ̃4. JCC1683c in JB2.1 was run in parallel as a negative control. Accordingly, a clonal starter culture of JCC1683c and JCC1685 was grown up for 5 days at 37° C. at 150 rpm in 2% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron H (Infors) shaking photoincubator in A+supplemented with 100 μg/ml spectinomycin. At this point, each starter culture was used to inoculate duplicate 30 ml JB2.1 medium flask cultures supplemented with 400 μg/ml spectinomycin; in addition, the JCC1685 starter culture was used to inoculate duplicate 30 ml JB2.1 medium plus 6 mM urea flask cultures supplemented with 400 μg/ml spectinomycin. The six cultures were then grown for 14 days at 37° C. at 150 rpm in 2% CO2-enriched air at  ̃100 μmol photons m−2 s−1 in a Multitron II (Infors) shaking photoincubator. Intracellular n-alkanes as a percentage of DCW were determined exactly as described in Example 11; data are summarized in Table 9. Consistent with the urea repressibility of P(nir07), n-alkanes as a percentage of JCC185 DCW were significantly higher in the absence of urea ( ̃0.59%) compared to in the presence of urea ( ̃0.15%). This likely explained the relatively low final OD730 of the no-urea cultures.







TABLE 9







Table 9 n-Pentadecane and n-heptadecane quantitated by GC-FID in acetone cell pellet


extractants of JCC1683c and JCC1685 as a function of urea in the growth medium.


















C27-normalized
C15 as % of
C17 as % of
n-alkanes
(C15 + C17)/(C13 + C14 +






extraction/injection
DCW (95%)
DCW (95%
as % of
C15 + C16 + C17)
C15/(C15 + C17)


Strain
Medium
OD730
efficiency
CI %)
CI %)
DCW
Mass %
Mass %





JCC1683c #1
JB2.1
9.5
101%
nd
nd
na
na
na


JCC1683c #2
JB2.1
9.5
101%
nd
nd
na
na
na


JCC1685 #1
JB2.1 +
7.4
102%
0.076% (7.1%)
0.067% (1.5%)
0.14%
 100%
53.2%



6 mM


JCC1685 #2
JB2.1 +
6.4
102%
0.090% (3.3%)
0.051% (2.3%)
0.15%
94.6%
63.9%



6 mM


JCC1685 #1
JB2.1
1.2
101%
 0.42% (1.4%)
 0.14% (1.1%)
0.57%
97.9%
74.9%


JCC1685 #2
JB2.1
3.3
102%
 0.49% (1.6%)
 0.11% (1.6%)
0.60%
 100%
81.4%





n-Octadecane was not detected in any of the samples; nd: not detected, na: not applicable.






A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. All publications, patents and other references mentioned herein are hereby incorporated by reference in their entirety.










INFORMAL SEQUENCE LISTING















SEQ ID NO: 1


ThermoSynechococcus elongatus BP-1; tll1312 (AAR)


nucleic acid sequence


ATGTTTGGATTAATTGGTCATCTGACGAGTCTGGAGCACGCCCAAGCCGTTGCCCATCAGTTGGG


TTACCCCGAATATGCCGATCAAGGCTTGGAATTTTGGTGTATGGCACCGCCGCAGATCGTCGATG


AGATTACGGTGACGAGCGTAACGGGCAAAACTATCTATGGCAAATACGTTGAGTCCTGCTTTTTA


CCAGAGATGCTGGCCAACCAGCGGGTGAAGGCAGCGACTCGCAAAGTTATTAACGCCATGGCCCA


TGCCCAAAAGCACAACATTGACATTACGGCCTTGGGGGGCTTCTCCTCGATCATCTTTGAGAACT


TTGATCTGGAGAAAATGTCCCACATTCGCAACATTGAACTGGACTTTCGCCGCTTTACAACGGGG


AATACCCATACCGCCTATATCATCTGCCAACAAATTGAGCAGGCGGCGCCCCAAGTGGGGATTGA


TTTGCGGCAGGCAACCGTGGCTGTTTGTGGGGCTACGGGGGATATTGGTAGTGCCGTCTGCCGTT


GGTTGAATACCTGTTTAGATGTGCAAGATCTCTTACTCGTAGCACGGAATCGCGATCGCCTGCTG


GAGCTACAGGCGGAATTGGGACGGGGGAAAATCCTCGACTTGATGGAGGCGCTGCCCCTTGCCGA


TATTGTGGTTTGGGTGGCCAGTATGCCCAAGGGAGTTGAGCTGAGCATTGAGCAGTTAAAACGCC


CCTCCCTGATGATTGATGGTGGTTATCCCAAAAATATGGCCACCAAAATTCAGCACCCCCAGATT


CATGTTCTCAATGGTGGCATTGTCGAGCATGCCCTCGACATTGACTGGAAAATTATGGAAATTGT


GAATATGGATGTGCCCTCGCGGCAGATGTTTGCCTGTTTTGCAGAGGCTATGCTTTTAGAGTTCG


AGGGCTGGCACACCAATTTCTCTTGGGGACGCAATCAAATCACTGTGGAAAAGATGCAGCAAATT


GGTGAGGTCTCCCGTAAACATGGATTTCAGCCACTACTGTTGAATCCTCAGTAA (SEQ ID NO: 1)


 


SEQ ID NO: 2


ThermoSynechococcus elongatus BP-1 tll1312 (AAR)


amino acid sequence


MFGLIGHLTSLEHAQAVAHQLGYPEYADQGLEFWCMAPPQIVDEITVTSVTGKTIYGKYVESCFL


PEMLANQRVKAATRKVINAMAHAQKHNIDITALGGFSSIIFENFDLEKMSHIRNIELDFRRFTTG


NTHTAYIICQQIEQAAPQVGIDLRQATVAVCGATGDIGSAVCRWLNTCLDVQDLLLVARNRDRLL


ELQAELGRGKILDLMEALPLADIVVWVASMPKGVELSIEQLKRPSLMIDGGYPKNMATKIQHPQI


HVLNGGIVEHALDIDWKIMEIVNMDVPSRQMFACFAEAMLLEFEGWHTNFSWGRNQITVEKMQQI


GEVSRKHGFQPLLLNPQ (SEQ ID NO: 2)


 


SEQ ID NO: 3


ThermoSynechococcus elongatus BP-1 tll1313 (ADM)


nucleic acid sequence


ATGACAACGGCTACCGCTACACCTGTTTTGGACTACCATAGCGATCGCTACAAGGATGCCTACAG


CCGCATTAACGCCATTGTCATTGAAGGTGAACAGGAAGCTCACGATAACTATATCGATTTAGCCA


AGCTGCTGCCACAACACCAAGAGGAACTCACCCGCCTTGCCAAGATGGAAGCTCGCCACAAAAAG


GGGTTTGAGGCCTGTGGTCGCAACCTGAGCGTAACGCCAGATATGGAATTTGCCAAAGCCTTCTT


TGAAAAACTGCGCGCTAACTTTCAGAGGGCTCTGGCGGAGGGAAAAACTGCGACTTGTCTTCTGA


TTCAAGCTTTGATCATCGAATCCTTTGCGATCGCGGCCTACAACATCTACATCCCAATGGCGGAT


CCTTTCGCCCGTAAAATTACTGAGAGTGTTGTTAAGGACGAATACAGCCACCTCAACTTTGGCGA


AATCTGGCTCAAGGAACACTTTGAAAGCGTCAAAGGAGAGCTCGAAGAAGCCAATCGCGCCAATT


TACCCTTGGTCTGGAAAATGCTCAACCAAGTGGAAGCAGATGCCAAAGTGCTCGGCATGGAAAAA


GATGCCCTTGTGGAAGACTTCATGATTCAGTACAGTGGTGCCCTAGAAAATATCGGCTTTACCAC


CCGCGAAATTATGAAGATGTCAGTTTATGGCCTCACTGGGGCATAA (SEQ ID NO: 3)


 


SEQ ID NO: 4


ThermoSynechococcus elongatus BP-1 tll1313 (ADM)


amino acid sequence


MTTATATPVLDYHSDRYKDAYSRINAIVIEGEQEAHDNYIDLAKLLPQHQEELTRLAKMEARHKK


GFEACGRNLSVTPDMEFAKAFFEKLRANFQRALAEGKTATCLLIQALTIESFAIAAYNIYIPMAD


PFARKITESVVKDEYSHLNFGEIWLKEHFESVKGELEEANRANLPLVWKMLNQVEADAKVLGMEK


DALVEDFMIQYSGALENIGFTTREIMKMSVYGLTGA (SEQ ID NO: 4)


 


SEQ ID NO: 5


Synechococcus elongatus PCC 7942; SYNPCC7942_1594 (AAR)


nucleic acid sequence


ATGTTCGGTCTTATCGGTCATCTCACCAGTTTGGAGCAGGCCCGCGACGTTTCTCGCAGGATGG


GCTACGACGAATACGCCGATCAAGGATTGGAGTTTTGGAGTAGCGCTCCTCCTCAAATCGTTGA


TGAAATCACAGTCACCAGTGCCACAGGCAAGGTGATTCACGGTCGCTACATCGAATCGTGTTTC


TTGCCGGAAATGCTGGCGGCGCGCCGCTTCAAAACAGCCACGCGCAAAGTTCTCAATGCCATGT


CCCATGCCCAAAAACACGGCATCGACATCTCGGCCTTGGGGGGCTTTACCTCGATTATTTTCGA


GAATTTCGATTTGGCCAGTTTGCGGCAAGTGCGCGACACTACCTTGGAGTTTGAACGGTTCACC


ACCGGCAATACTCACACGGCCTACGTAATCTGTAGACAGGTGGAAGCCGCTGCTAAAACGCTGG


GCATCGACATTACCCAAGCGACAGTAGCGGTTGTCGGCGCGACTGGCGATATCGGTAGCGCTGT


CTGCCGCTGGCTCGACCTCAAACTGGGTGTCGGTGATTTGATCCTGACGGCGCGCAATCAGGAG


CGTTTGGATAACCTGCAGGCTGAACTCGGCCGGGGCAAGATTCTGCCCTTGGAAGCCGCTCTGC


CGGAAGCTGACTTTATCGTGTGGGTCGCCAGTATGCCTCAGGGCGTAGTGATCGACCCAGCAAC


CCTGAAGCAACCCTGCGTCCTAATCGACGGGGGCTACCCCAAAAACTTGGGCAGCAAAGTCCAA


GGTGAGGGCATCTATGTCCTCAATGGCGGGGTAGTTGAACATTGCTTCGACATCGACTGGCAGA


TCATGTCCGCTGCAGAGATGGCGCGGCCCGAGCGCCAGATGTTTGCCTGCTTTGCCGAGGCGAT


GCTCTTGGAATTTGAAGGCTGGCATACTAACTTCTCCTGGGGCCGCAACCAAATCACGATCGAG


AAGATGGAAGCGATCGGTGAGGCATCGGTGCGCCACGGCTTCCAACCCTTGGCATTGGCAATTT


GA (SEQ ID NO: 5)


 


SEQ ID NO: 6


Synechococcus elongatus PCC 7942; SYNPCC7942_1594 (AAR)


amino acid sequence


MFGLIGHLTSLEQARDVSRRMGYDEYADQGLEFWSSAPPQIVDEITVTSATGKVIHGRYIESCFL


PEMLAARRFKTATRKVLNAMSHAQKHGIDISALGGFTSIIFENFDLASLRQVRDTTLEFERFTTG


NTHTAYVICRQVEAAAKTLGIDITQATVAVVGATGDIGSAVCRWLDLKLGVGDLILTARNQERLD


NLQAELGRGKILPLEAALPEADFIVWVASMPQGVVIDPATLKQPCVLIDGGYPKNLGSKVQGEGI


YVLNGGVVEHCFDIDWQIMSAAEMARPERQMFACFAEAMLLEFEGWHTNFSWGRNQITIEKMEAI


GEASVRHGFQPLALAI (SEQ ID NO: 6)


 


SEQ ID NO: 7


Synechococcus elongatus PCC 7942; SYNPCC7942_1593 (ADM)


nucleic acid sequence


ATGCCGCAGCTTGAAGCCAGCCTTGAACTGGACTTTCAAAGCGAGTCCTACAAAGACGCTTACAG


CCGCATCAACGCGATCGTGATTGAAGGCGAACAAGAGGCGTTCGACAACTACAATCGCCTTGCTG


AGATGCTGCCCGACCAGCGGGATGAGCTTCACAAGCTAGCCAAGATGGAACAGCGCCACATGAAA


GGCTTTATGGCCTGTGGCAAAAATCTCTCCGTCACTCCTGACATGGGTTTTGCCCAGAAATTTTT


CGAGCGCTTGCACGAGAACTTCAAAGCGGCGGCTGCGGAAGGCAAGGTCGTCACCTGCCTACTGA


TTCAATCGCTAATCATCGAGTGCTTTGCGATCGCGGCTTACAACATCTACATCCCAGTGGCGGAT


GCTTTTGCCCGCAAAATCACGGAGGGGGTCGTGCGCGACGAATACCTGCACCGCAACTTCGGTGA


AGAGTGGCTGAAGGCGAATTTTGATGCTTCCAAAGCCGAACTGGAAGAAGCCAATCGTCAGAACC


TGCCCTTGGTTTGGCTAATGCTCAACGAAGTGGCCGATGATGCTCGCGAACTCGGGATGGAGCGT


GAGTCGCTCGTCGAGGACTTTATGATTGCCTACGGTGAAGCTCTGGAAAACATCGGCTTCACAAC


GCGCGAAATCATGCGTATGTCCGCCTATGGCCTTGCGGCCGTTTGA (SEQ ID NO: 7)


 


SEQ ID NO: 8


Synechococcus elongatus PCC 7942; SYNPCC7942_1593 (ADM)


amino acid sequence


MPQLEASLELDFQSESYKDAYSRINAIVIEGEQEAFDNYNRLAEMLPDQRDELHKLAKMEQRHMK


GFMACGKNLSVTPDMGFAQKFFERLHENFKAAAAEGKVVTCLLIQSLIIECFAIAAYNIYIPVAD


AFARKITEGVVRDEYLHRNFGEEWLKANFDASKAELEEANRQNLPLVWLMLNEVADDARELGMER


ESLVEDFMIAYGEALENIGFTTREIMRMSAYGLAAV (SEQ ID NO: 8)


 


SEQ ID NO: 9


Prochlorococcus marinus MIT9312 PMT9312_0533 (AAR)


optimized nucleic acid sequence


ATGTTTGGTCTGATTGGTCATAGCACCAGCTTTGAGGACGCAAAGCGCAAGGCGAGCCTGCTGGG


TTTCGACCACATCGCGGATGGCGATCTGGATGTGTGGTGTACCGCACCGCCGCAACTGGTTGAAA


ACGTGGAAGTCAAAAGCGCGACGGGTATCAGCATTGAAGGTAGCTATATCGATAGCTGCTTCGTG


CCGGAGATGCTGAGCCGCTTCAAGACCGCGCGTCGTAAAGTTCTGAATGCAATGGAGCTGGCGCA


GAAAAAGGGTATCAATATCACTGCCCTGGGTGGCTTTACCTCCATTATCTTTGAGAACTTCAACC


TGTTGCAGCACAAGCAAATCCGTAATACCAGCCTGGAGTGGGAGCGTTTCACCACGGGTAACACG


CACACGGCATGGGTGATTTGTCGTCAGCTGGAGATCAACGCACCGCGCATTGGCATCGACCTGAA


AACTGCAACGGTCGCTGTTATCGGCGCGACCGGCGATATTGGTAGCGCGGTGTGTCGCTGGCTGG


TCAATAAGACCGGCATTAGCGAACTGCTGATGGTCGCTCGCCAACAACAGCCACTGACCCTGCTG


CAAAAAGAACTGGACGGTGGCACCATCAAGAGCCTGGATGAAGCCCTGCCGCAGGCGGATATTGT


CGTGTGGGTTGCTTCGATGCCTAAGACGATCGAAATTGAGATTGAAAACCTGAAAAAGCCGTGCC


TGATGATCGACGGTGGCTACCCGAAGAATCTGGACGAGAAATTCAAAGGCAAAAACATTCACGTG


TTGAAGGGTGGTATCGTCGAGTTTTTCAACGACATTGGCTGGAACATGATGGAGTTGGCGGAGAT


GCAAAACCCGCAGCGTGAGATGTTTGCGTGCTTCGCCGAAGCTATGATTCTGGAGTTTGAGAAAT


GCCATACCAACTTTAGCTGGGGCCGTAACAATATCAGCTTGGAGAAGATGGAGTTCATCGGTGCT


GCATCTCTGAAGCACGGTTTCAGCGCGATCGGTCTGGATAAACAGCCGAAAGTCTTGACCGTTTG


A (SEQ ID NO: 9)


 


SEQ ID NO: 10


Prochlorococcus marinus MIT9312 PMT9312_0533 (AAR)


protein sequence


MFGLIGHSTSFEDAKRKASLLGFDHIADGDLDVWCTAPPQLVENVEVKSATGISIEGSYIDSCFV


PEMLSRFKTARRKVLNAMELAQKKGINITALGGFTSIIFENFNLLQHKQIRNTSLEWERFTTGNT


HTAWVICRQLEINAPRIGIDLKTATVAVIGATGDIGSAVCRWLVNKTGISELLMVARQQQPLTLL


QKELDGGTIKSLDEALPQADIVVWVASMPKTIEIEIENLKKPCLMIDGGYPKNLDEKFKGKNIHV


LKGGIVEFFNDIGWNMMELAEMQNPQREMFACFAEAMILEEKCHTNFSWGRNNISLEKMEFIGAA


SLKHGFSAIGLDKQPKVLTV (SEQ ID NO: 10)


 


SEQ ID NO: 11


Prochlorococcus marinus MIT9312 PMT9312_0532 (ADM)


optimized nucleic acid sequence


ATGCACAATGAATTGAAAATCACGGATATGCAAACGCTGGAAACCAACACCAAGACGACCGAAGA


GTCTATTGACACCAATAGCCTGAACCTGCCGGACTTTACTACCGACAGCTACAAGGATGCCTATT


CTCGCATTAACGCCATCGTTATTGAGGGCGAACAGGAAGCTCATGACAATTACATCTCCATCGCA


ACGCTGATCCCGAATGAGCTGGAAGAGCTGACGAAGCTGGCACGTATGGAGCTGAAACACAAGAA


AGGTTTTACTGCGTGCGGTCGTAATCTGGGTGTGGACGCAGACATGGTTTTCGCGAAAAAGTTCT


TCAGCAAACTGCACGGCAATTTCCAAATCGCGCTGGAAAAAGGTAACCTGACCACCTGCTTGCTG


ATCCAAGCGATTCTGATCGAAGCATTTGCGATTTCCGCGTACAATGTTTACATCCGTGTGGCCGA


CCCATTTGCCAAAAAGATTACCGAGGGTGTTGTCAAAGACGAGTATCTGCATCTGAACTATGGTC


AGGAGTGGCTGAAAAAGAATCTGTCCACGTGTAAAGAAGAGCTGATGGAGGCCAACAAGGTCAAT


CTGCCGCTGATTAAGAAAATGCTGGACGAAGTGGCAGAAGATGCGAGCGTTTTGGCGATGGATCG


TGAAGAGTTGATGGAAGAGTTCATGATTGCGTACCAGGATACCCTGTTGGAGATTGGCCTGGATA


ATCGCGAAATTGCCCGTATGGCGATGGCGGCCATTGTTTAG (SEQ ID NO: 11)


 


SEQ ID NO: 12


Prochlorococcus marinus MIT9312 PMT9312_0532 (ADM)


protein sequence


MHNELKITDMQTLETNTKTTEESIDTNSLNLPDFTTDSYKDAYSRINAIVIEGEQEAHDNYISIA


TLIPNELEELTKLARMELKHKKGFTACGRNLGVDADMVFAKKFFSKLHGNFQIALEKGNLTTCLL


IQAILIEAFAISAYNVYIRVADPFAKKITEGVVKDEYLHLNYGQEWLKKNLSTCKEELMEANKVN


LPLIKKMLDEVAEDASVLAMDREELMEEFMIAYQDTLLEIGLDNREIARMAMAAIV (SEQ ID NO: 12)


 


SEQ ID NO: 13


Prochlorococcus marinus MIT9312 PMT9312_0533 (AAR)


nucleic acid sequence


ATGTTTGGGTTAATAGGCCACTCAACTAGTTTTGAAGATGCAAAAAGAAAAGCTTCATTACTAGG


CTTTGATCATATTGCTGATGGTGATCTAGATGTTTGGTGTACAGCCCCTCCTCAATTGGTTGAAA


ATGTAGAAGTTAAGAGTGCTACTGGAATATCTATTGAAGGTTCTTATATAGATTCTTGCTTTGTT


CCTGAAATGCTTTCTAGGTTTAAAACCGCAAGAAGAAAAGTATTAAATGCTATGGAATTAGCTCA


GAAAAAAGGGATTAACATTACGGCTTTAGGAGGATTTACTTCTATTATTTTCGAAAATTTTAATC


TTCTTCAACATAAACAAATTAGAAATACTTCATTAGAGTGGGAAAGGTTTACTACAGGTAATACA


CACACTGCCTGGGTTATTTGTAGGCAACTAGAAATAAATGCTCCTCGCATAGGGATAGATCTTAA


AACTGCAACTGTTGCTGTTATTGGTGCTACAGGTGATATAGGAAGTGCTGTTTGTAGGTGGCTTG


TCAATAAAACTGGTATTTCAGAACTTCTTATGGTGGCTAGACAACAACAACCATTAACTCTATTA


CAGAAAGAATTAGATGGTGGCACTATAAAAAGTTTAGATGAAGCATTGCCTCAAGCGGATATTGT


TGTATGGGTTGCAAGTATGCCTAAAACGATTGAAATTGAAATTGAAAACTTAAAAAAACCATGTT


TAATGATTGATGGTGGATACCCTAAAAATCTTGATGAGAAATTTAAAGGTAAAAATATTCATGTT


TTAAAAGGAGGTATAGTAGAGTTTTTCAATGATATTGGCTGGAATATGATGGAACTTGCAGAAAT


GCAGAACCCTCAGAGAGAGATGTTTGCTTGCTTTGCAGAAGCTATGATTTTAGAATTTGAAAAGT


GTCATACCAACTTTAGTTGGGGAAGGAATAACATTTCTCTTGAAAAAATGGAATTTATTGGAGCA


GCTTCTTTGAAACATGGTTTTTCTGCGATTGGACTTGATAAACAGCCTAAAGTATTGACTGTTTG


A (SEQ ID NO: 13)


 


SEQ ID NO: 14


Prochlorococcus marinus MIT9312 PMT9312_0532 (ADM)


nucleic acid sequence


ATGCATAATGAGCTAAAGATTACTGACATGCAAACTCTAGAAACAAATACAAAAACTACTGAAGA


ATCCATAGACACGAATTCTTTGAATCTTCCCGACTTTACAACAGATTCCTATAAGGATGCATATA


GCAGAATAAATGCAATTGTTATAGAGGGAGAGCAAGAGGCTCATGATAATTACATTTCAATAGCA


ACGTTAATACCAAATGAGTTAGAAGAATTAACTAAGTTGGCGAGAATGGAACTCAAGCATAAAAA


AGGATTTACTGCTTGTGGAAGAAATTTAGGAGTAGATGCTGATATGGTATTCGCAAAAAAATTCT


TTTCTAAATTGCATGGTAATTTTCAAATTGCTTTAGAAAAAGGAAATTTAACAACTTGTCTTCTG


ATACAAGCTATTTTAATTGAAGCTTTTGCTATATCTGCTTATAACGTTTACATAAGAGTTGCTGA


TCCTTTTGCAAAAAAAATAACAGAGGGAGTGGTTAAAGATGAATATCTCCATCTAAATTACGGCC


AAGAGTGGCTTAAAAAGAATTTATCTACTTGTAAAGAAGAATTAATGGAAGCCAATAAGGTTAAC


CTTCCCTTAATTAAAAAGATGTTAGATGAAGTAGCAGAAGATGCATCAGTTTTGGCTATGGATAG


AGAAGAGTTAATGGAAGAATTTATGATTGCTTACCAAGACACTCTTCTAGAAATAGGTCTTGATA


ATAGAGAAATTGCAAGAATGGCTATGGCAGCGATTGTTTAA (SEQ ID NO: 14)


 


SEQ ID NO: 15


plasmid JB823








1st underlined sequence
Upstream homology region for pAQ1


1st double-underlined lowercase 
NotI site for promoter swapping


sequence



1st italic sequence
aphII promoter


2nd double-underlined lowercase 
NdeI site for promoter swapping


sequence



1st bold sequence
SYNPCC7942_1593 (adm) coding sequence


1st lower case sequence
Intergenic sequence


2nd bold sequence
SYNPCC7942_1594 (aar) coding sequence


2nd italic sequence
aadA coding sequence; specR selection 



marker


2nd underlined sequence
Downstream homology region for pAQ1


2nd lowercase sequence
Vector backbone







GTCAGCAAGCTCTGGAATTTCCCGATTCTCTGATGGGAGATCCAAAAATTCTCGCAGTCCCTCA


ATCACGATATCGGTCTTGGATCGCCCTGTAGCTTCCGACAACTGCTCAATTTTTTCGAGCATCT


CTACCGGGCATCGGAATGAAATTAACGGTGTTTTAGCCATGTGTTATACAGTGTTTACAACTTG


ACTAACAAATACCTGCTAGTGTATACATATTGTATTGCAATGTATACGCTATTTTCACTGCTGT


CTTTAATGGGGATTATCGCAAGCAAGTAAAAAAGCCTGAAAACCCCAATAGGTAAGGGATTCCG


AGCTTACTCGATAATTATCACCTTTGAGCGCCCCTAGGAGGAGGCGAAAAGCTATGTCTGACAA


GGGGTTTGACCCCTGAAGTCGTTGCGCGAGCATTAAGGTCTGCGGATAGCCCATAACATACTTT


TGTTGAACTTGTGCGCTTTTATCAACCCCTTAAGGGCTTGGGAGCGTTTTATgcggccgcGGGG


GGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATAT


ATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTcatatgCCGCAG


CTTGAAGCCAGCCTTGAACTGGACTTTCAAAGCGAGTCCTACAAAGACGCTTACAGCCGCATCA


ACGCGATCGTGATTGAAGGCGAACAAGAGGCGTTCGACAACTACAATCGCCTTGCTGAGATGCT


GCCCGACCAGCGGGATGAGCTTCACAAGCTAGCCAAGATGGAACAGCGCCACATGAAAGGCTTT


ATGGCCTGTGGCAAAAATCTCTCCGTCACTCCTGACATGGGTTTTGCCCAGAAATTTTTCGAGC


GCTTGCACGAGAACTTCAAAGCGGCGGCTGCGGAAGGCAAGGTCGTCACCTGCCTACTGATTCA


ATCGCTAATCATCGAGTGCTTTGCGATCGCGGCTTACAACATCTACATCCCAGTGGCGGATGCT


TTTGCCCGCAAAATCACGGAGGGGGTCGTGCGCGACGAATACCTGCACCGCAACTTCGGTGAAG


AGTGGCTGAAGGCGAATTTTGATGCTTCCAAAGCCGAACTGGAAGAAGCCAATCGTCAGAACCT


GCCCTTGGTTTGGCTAATGCTCAACGAAGTGGCCGATGATGCTCGCGAACTCGGGATGGAGCGT


GAGTCGCTCGTCGAGGACTTTATGATTGCCTACGGTGAAGCTCTGGAAAACATCGGCTTCACAA


CGCGCGAAATCATGCGTATGTCCGCCTATGGCCTTGCGGCCGTTTGAtccaggaaatctgaATG


TTCGGTCTTATCGGTCATCTCACCAGTTTGGAGCAGGCCCGCGACGTTTCTCGCAGGATGGGCT


ACGACGAATACGCCGATCAAGGATTGGAGTTTTGGAGTAGCGCTCCTCCTCAAATCGTTGATGA


AATCACAGTCACCAGTGCCACAGGCAAGGTGATTCACGGTCGCTACATCGAATCGTGTTTCTTG


CCGGAAATGCTGGCGGCGCGCCGCTTCAAAACAGCCACGCGCAAAGTTCTCAATGCCATGTCCC


ATGCCCAAAAACACGGCATCGACATCTCGGCCTTGGGGGGCTTTACCTCGATTATTTTCGAGAA


TTTCGATTTGGCCAGTTTGCGGCAAGTGCGCGACACTACCTTGGAGTTTGAACGGTTCACCACC


GGCAATACTCACACGGCCTACGTAATCTGTAGACAGGTGGAAGCCGCTGCTAAAACGCTGGGCA


TCGACATTACCCAAGCGACAGTAGCGGTTGTCGGCGCGACTGGCGATATCGGTAGCGCTGTCTG


CCGCTGGCTCGACCTCAAACTGGGTGTCGGTGATTTGATCCTGACGGCGCGCAATCAGGAGCGT


TTGGATAACCTGCAGGCTGAACTCGGCCGGGGCAAGATTCTGCCCTTGGAAGCCGCTCTGCCGG


AAGCTGACTTTATCGTGTGGGTCGCCAGTATGCCTCAGGGCGTAGTGATCGACCCAGCAACCCT


GAAGCAACCCTGCGTCCTAATCGACGGGGGCTACCCCAAAAACTTGGGCAGCAAAGTCCAAGGT


GAGGGCATCTATGTCCTCAATGGCGGGGTAGTTGAACATTGCTTCGACATCGACTGGCAGATCA


TGTCCGCTGCAGAGATGGCGCGGCCCGAGCGCCAGATGTTTGCCTGCTTTGCCGAGGCGATGCT


CTTGGAATTTGAAGGCTGGCATACTAACTTCTCCTGGGGCCGCAACCAAATCACGATCGAGAAG


ATGGAAGCGATCGGTGAGGCATCGGTGCGCCACGGCTTCCAACCCTTGGCATTGGCAATTTGAG


GTCTGTGAATTCGGTTTTCCGTCCTGTCTTGATTTTCAAGCAAACAATGCCTCCGATTTCTAAT


CGGAGGCATTTGTTTTTGTTTATTGCAAAAACAAAAAATATTGTTACAAATTTTTACAGGCTAT


TAAGCCTACCGTCATAAATAATTTGCCATTTACTAGTTTTTAATTAACCAGAACCTTGACCGAA


CGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTAC


AGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATG


GAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAA


GCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCG


AACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAG


TGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATC


AACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCA


CCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGG


AGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTG


GCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAAC


TCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAA


CTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTAC


AGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGC


CGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCG


CTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAG


GTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACT


CAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTT


ATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGAGGCGCGCCAC


GAGTGCGGGGAAATTTCGGGGGCGATCGCCCCTATATCGCAAAAAGGAGTTACCCCATCAGAGC


TATAGTCGAGAAGAAAACCATCATTCACTCAACAAGGCTATGTCAGAAGAGAAACTAGACCGGA


TCGAAGCAGCCCTAGAGCAATTGGATAAGGATGTGCAAACGCTCCAAACAGAGCTTCAGCAATC


CCAAAAATGGCAGGACAGGACATGGGATGTTGTGAAGTGGGTAGGCGGAATCTCAGCGGGCCTA


GCGGTGAGCGCTTCCATTGCCCTGTTCGGGTTGGTCTTTAGATTTTCTGTTTCCCTGCCATAAA


AGCACATTCTTATAAGTCATACTTGTTTACATCAAGGAACAAAAACGGCATTGTGCCTTGCAAG


GCACAATGTCTTTCTCTTATGCACAGATGGGGACTGGAAACCACACGCACAATTCCCTTAAAAA


GCAACCGCAAAAAATAACCATCAAAATAAAACTGGACAAATTCTCATGTGggccggccaaaatg


aagtgaagttcctatactttctagagaataggaacttctatagtgagtcgaataagggcgacac


aaaatttattctaaatgcataataaatactgataacatcttatagtttgtattatattttgtat


tatcgttgacatgtataattttgatatcaaaaactgattttccctttattattttcgagattta


ttttcttaattctctttaacaaactagaaatattgtatatacaaaaaatcataaataatagatg


aatagtttaattataggtgttcatcaatcgaaaaagcaacgtatcttatttaaagtgcgttgct


tttttctcatttataaggttaaataattctcatatatcaagcaaagtgacaggcgcccttaaat


attctgacaaatgctctttccctaaactccccccataaaaaaacccgccgaagcgggtttttac


gttatttgcggattaacgattactcgttatcagaaccgcccagggggcccgagcttaagactgg


ccgtcgttttacaacacagaaagagtttgtagaaacgcaaaaaggccatccgtcaggggccttc


tgcttagtttgatgcctggcagttccctactctcgccttccgcttcctcgctcactgactcgct


gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcc


acagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc


gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa


tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct


ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc


tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt


cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatcc


ggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg


gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgggctaa


ctacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgga


aaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt


gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg


gtctgacgctcagtggaacgacgcgcgcgtaactcacgttaagggattttggtcatgagcttgc


gccgtcccgtcaagtcagcgtaatgctctgcttttaccaatgcttaatcagtgaggcacctatc


tcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacga


tacgggagggcttaccatctggccccagcgctgcgatgataccgcgagaaccacgctcaccggc


tccggatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaact


ttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagtta


atagtttgcgcaacgttgttgccatcgctacaggcatcgtggtgtcacgctcgtcgtttggtat


ggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaa


aaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcac


tcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgt


gactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgc


ccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaa


aacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacc


cactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaa


acaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatat


tcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatt


tgaatgtatttagaaaaataaacaaataggggtcagtgttacaaccaattaaccaattctgaac


attatcgcgagcccatttatacctgaatatggctcataacaccccttgtttgcctggcggcagt


agcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggta


gtgtggggactccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagt


cgaaagactgggcctttcgcccgggctaattatggggtgtcgcccttattcgactctatagtga


agttcctattctctagaaagtataggaacttctgaagtggggcctgcagg (SEQ ID NO: 15)


 


SEQ ID NO: 16


plasmid pJB855








1st bold sequence
SYNPCC7942_1593 coding sequence


2nd bold sequence
SYNPCC7942_1594 coding sequence


Italic sequence
aadA spectinomycin selection marker (reverse 



complement)







GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGAT


ATACCATGGGCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGAATTCGAGCTCGGCGCGC


CTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACA


CGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCA


TCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGCCGCAGCTTGAAGCCAGCCTTGA


ACTGGACTTTCAAAGCGAGTCCTACAAAGACGCTTACAGCCGCATCAACGCGATCGTGATTGAAG


GCGAACAAGAGGCGTTCGACAACTACAATCGCCTTGCTGAGATGCTGCCCGACCAGCGGGATGAG


CTTCACAAGCTAGCCAAGATGGAACAGCGCCACATGAAAGGCTTTATGGCCTGTGGCAAAAATCT


CTCCGTCACTCCTGACATGGGTTTTGCCCAGAAATTTTTCGAGCGCTTGCACGAGAACTTCAAAG


CGGCGGCTGCGGAAGGCAAGGTCGTCACCTGCCTACTGATTCAATCGCTAATCATCGAGTGCTTT


GCGATCGCGGCTTACAACATCTACATCCCAGTGGCGGATGCTTTTGCCCGCAAAATCACGGAGGG


GGTCGTGCGCGACGAATACCTGCACCGCAACTTCGGTGAAGAGTGGCTGAAGGCGAATTTTGATG


CTTCCAAAGCCGAACTGGAAGAAGCCAATCGTCAGAACCTGCCCTTGGTTTGGCTAATGCTCAAC


GAAGTGGCCGATGATGCTCGCGAACTCGGGATGGAGCGTGAGTCGCTCGTCGAGGACTTTATGAT


TGCCTACGGTGAAGCTCTGGAAAACATCGGCTTCACAACGCGCGAAATCATGCGTATGTCCGCCT


ATGGCCTTGCGGCCGTTTGATCCAGGAAATCTGAATGTTCGGTCTTATCGGTCATCTCACCAGTT


TGGAGCAGGCCCGCGACGTTTCTCGCAGGATGGGCTACGACGAATACGCCGATCAAGGATTGGAG


TTTTGGAGTAGCGCTCCTCCTCAAATCGTTGATGAAATCACAGTCACCAGTGCCACAGGCAAGGT


GATTCACGGTCGCTACATCGAATCGTGTTTCTTGCCGGAAATGCTGGCGGCGCGCCGCTTCAAAA


CAGCCACGCGCAAAGTTCTCAATGCCATGTCCCATGCCCAAAAACACGGCATCGACATCTCGGCC


TTGGGGGGCTTTACCTCGATTATTTTCGAGAATTTCGATTTGGCCAGTTTGCGGCAAGTGCGCGA


CACTACCTTGGAGTTTGAACGGTTCACCACCGGCAATACTCACACGGCCTACGTAATCTGTAGAC


AGGTGGAAGCCGCTGCTAAAACGCTGGGCATCGACATTACCCAAGCGACAGTAGCGGTTGTCGGC


GCGACTGGCGATATCGGTAGCGCTGTCTGCCGCTGGCTCGACCTCAAACTGGGTGTCGGTGATTT


GATCCTGACGGCGCGCAATCAGGAGCGTTTGGATAACCTGCAGGCTGAACTCGGCCGGGGCAAGA


TTCTGCCCTTGGAAGCCGCTCTGCCGGAAGCTGACTTTATCGTGTGGGTCGCCAGTATGCCTCAG


GGCGTAGTGATCGACCCAGCAACCCTGAAGCAACCCTGCGTCCTAATCGACGGGGGCTACCCCAA


AAACTTGGGCAGCAAAGTCCAAGGTGAGGGCATCTATGTCCTCAATGGCGGGGTAGTTGAACATT


GCTTCGACATCGACTGGCAGATCATGTCCGCTGCAGAGATGGCGCGGCCCGAGCGCCAGATGTTT


GCCTGCTTTGCCGAGGCGATGCTCTTGGAATTTGAAGGCTGGCATACTAACTTCTCCTGGGGCCG


CAACCAAATCACGATCGAGAAGATGGAAGCGATCGGTGAGGCATCGGTGCGCCACGGCTTCCAAC


CCTTGGCATTGGCAATTTGAGGTCTGTGAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCG


GTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCT


ACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGG


GGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCA


CACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCC


TGCCCTGAACCGACGACCGGGTCATCGTGGCCGGATCTTGCGGCCCCTCGGCTTGAACGAATTGT


TAGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGTAGTGGACAAATTCTTCCAACT


GATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATG


ACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGAT


TTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAG


CCCAGTCGGGCGGCGAGTTCCATAGCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCA


GGAACCGGATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGCTTT


TGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCAT


TGCGCTGCCATTCTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCG


TGCACAACAATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTC


CAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGCA


AATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGC


AACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGGC


GATCACCGCTTCCCTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC


TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGCTAGCTCACTCGGTCGC


TACGCTCCGGGCGTGAGACTGCGGCGGGCGCTGCGGACACATACAAAGTTACCCACAGATTCCGT


GGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGCCGCGCCGGTGGCGTTTTTCCATAG


GCTCCGCCCTCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGAGCCGTGA


GGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCC


GGCGGGTCGCTCCCTCTTGCGCTCTCCTGTTCCGACCCTGCCGTTTACCGGATACCTGTTCCGCC


TTTCTCCCTTACGGGAAGTGTGGCGCTTTCTCATAGCTCACACACTGGTATCTCGGCTCGGTGTA


GGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGACTGCTGCGCCTTAT


CCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATT


GGTAACTGGGAGTTCGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCA


AAGTCCGGCTACACTGGAAGGACAGATTTGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTT


AAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCTCCCCAGGTGGTTTTTTCGTTTACA


GGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACTGAACC


GCTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATA


AGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTC


TCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGC


TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC


GGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAAC


AGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCC


CAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTAT


CGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATT


GCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCAT


TTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAA


TTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT


GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGT


ACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACG


CCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATG


ATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCT


TCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGA


CAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTG


CCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT


TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGA


CACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTC


TCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTC


GACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA (SEQ ID NO: 16)


 


SEQ ID NO: 17


plasmid pJB947








1st underlined sequence
Upstream homology region for pAQ1


1st italic sequence
aphII promoter


1st bold sequence
PMT9312_0532 (adm) coding sequence


1st lower case sequence
Intergenic sequence


2nd bold sequence
PMT9312_0533 (aar)


2nd italic sequence
aadA coding sequence; spectinomycin selection 



marker


2nd underlined sequence
Downstream homology region for pAQ1


2nd lower case sequence
Vector backbone







GTCAGCAAGCTCTGGAATTTCCCGATTCTCTGATGGGAGATCCAAAAATTCTCGCAGTCCCTCAAT


CACGATATCGGTCTTGGATCGCCCTGTAGCTTCCGACAACTGCTCAATTTTTTCGAGCATCTCTAC


CGGGCATCGGAATGAAATTAACGGTGTTTTAGCCATGTGTTATACAGTGTTTACAACTTGACTAAC


AAATACCTGCTAGTGTATACATATTGTATTGCAATGTATACGCTATTTTCACTGCTGTCTTTAATG


GGGATTATCGCAAGCAAGTAAAAAAGCCTGAAAACCCCAATAGGTAAGGGATTCCGAGCTTACTCG


ATAATTATCACCTTTGAGCGCCCCTAGGAGGAGGCGAAAAGCTATGTCTGACAAGGGGTTTGACCC


CTGAAGTCGTTGCGCGAGCATTAAGGTCTGCGGATAGCCCATAACATACTTTTGTTGAACTTGTGC


GCTTTTATCAACCCCTTAAGGGCTTGGGAGCGTTTTATGCGGCCGCGGGGGGGGGGGGGAAAGCCA


CGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAA


ACTGTCTGCTTACATAAACAGTAATACAAGGGGTCATATGCACAATGAATTGAAAATCACGGATAT


GCAAACGCTGGAAACCAACACCAAGACGACCGAAGAGTCTATTGACACCAATAGCCTGAACCTGCC


GGACTTTACTACCGACAGCTACAAGGATGCCTATTCTCGCATTAACGCCATCGTTATTGAGGGCGA


ACAGGAAGCTCATGACAATTACATCTCCATCGCAACGCTGATCCCGAATGAGCTGGAAGAGCTGAC


GAAGCTGGCACGTATGGAGCTGAAACACAAGAAAGGTTTTACTGCGTGCGGTCGTAATCTGGGTGT


GGACGCAGACATGGTTTTCGCGAAAAAGTTCTTCAGCAAACTGCACGGCAATTTCCAAATCGCGCT


GGAAAAAGGTAACCTGACCACCTGCTTGCTGATCCAAGCGATTCTGATCGAAGCATTTGCGATTTC


CGCGTACAATGTTTACATCCGTGTGGCCGACCCATTTGCCAAAAAGATTACCGAGGGTGTTGTCAA


AGACGAGTATCTGCATCTGAACTATGGTCAGGAGTGGCTGAAAAAGAATCTGTCCACGTGTAAAGA


AGAGCTGATGGAGGCCAACAAGGTCAATCTGCCGCTGATTAAGAAAATGCTGGACGAAGTGGCAGA


AGATGCGAGCGTTTTGGCGATGGATCGTGAAGAGTTGATGGAAGAGTTCATGATTGCGTACCAGGA


TACCCTGTTGGAGATTGGCCTGGATAATCGCGAAATTGCCCGTATGGCGATGGCGGCCATTGTTTA


GtaatatttctaattaactaataaaggaagtctgaATGTTTGGTCTGATTGGTCATAGCACCAGCT


TTGAGGACGCAAAGCGCAAGGCGAGCCTGCTGGGTTTCGACCACATCGCGGATGGCGATCTGGATG


TGTGGTGTACCGCACCGCCGCAACTGGTTGAAAACGTGGAAGTCAAAAGCGCGACGGGTATCAGCA


TTGAAGGTAGCTATATCGATAGCTGCTTCGTGCCGGAGATGCTGAGCCGCTTCAAGACCGCGCGTC


GTAAAGTTCTGAATGCAATGGAGCTGGCGCAGAAAAAGGGTATCAATATCACTGCCCTGGGTGGCT


TTACCTCCATTATCTTTGAGAACTTCAACCTGTTGCAGCACAAGCAAATCCGTAATACCAGCCTGG


AGTGGGAGCGTTTCACCACGGGTAACACGCACACGGCATGGGTGATTTGTCGTCAGCTGGAGATCA


ACGCACCGCGCATTGGCATCGACCTGAAAACTGCAACGGTCGCTGTTATCGGCGCGACCGGCGATA


TTGGTAGCGCGGTGTGTCGCTGGCTGGTCAATAAGACCGGCATTAGCGAACTGCTGATGGTCGCTC


GCCAACAACAGCCACTGACCCTGCTGCAAAAAGAACTGGACGGTGGCACCATCAAGAGCCTGGATG


AAGCCCTGCCGCAGGCGGATATTGTCGTGTGGGTTGCTTCGATGCCTAAGACGATCGAAATTGAGA


TTGAAAACCTGAAAAAGCCGTGCCTGATGATCGACGGTGGCTACCCGAAGAATCTGGACGAGAAAT


TCAAAGGCAAAAACATTCACGTGTTGAAGGGTGGTATCGTCGAGTTTTTCAACGACATTGGCTGGA


ACATGATGGAGTTGGCGGAGATGCAAAACCCGCAGCGTGAGATGTTTGCGTGCTTCGCCGAAGCTA


TGATTCTGGAGTTTGAGAAATGCCATACCAACTTTAGCTGGGGCCGTAACAATATCAGCTTGGAGA


AGATGGAGTTCATCGGTGCTGCATCTCTGAAGCACGGTTTCAGCGCGATCGGTCTGGATAAACAGC


CGAAAGTCTTGACCGTTTGAaattGAATTCGGTTTTCCGTCCTGTCTTGATTTTCAAGCAAACAAT


GCCTCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATTGCAAAAACAAAAAATATTGTTACAAA


TTTTTACAGGCTATTAAGCCTACCGTCATAAATAATTTGCCATTTACTAGTTTTTAATTAACCAGA


ACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTT


TTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTG


ATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATG


AGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCAT


CTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACAC


AGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATC


AACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACC


ATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAA


TGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATC


TTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGAT


CCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCC


GACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACC


GGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAG


CCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCA


GATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGT


CTAACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAG


CACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAAT


AAGCCCTACACAAATTGGGAGATATATCATGAGGCGCGCCACGAGTGCGGGGAAATTTCGGGGGCG


ATCGCCCCTATATCGCAAAAAGGAGTTACCCCATCAGAGCTATAGTCGAGAAGAAAACCATCATTC


ACTCAACAAGGCTATGTCAGAAGAGAAACTAGACCGGATCGAAGCAGCCCTAGAGCAATTGGATAA


GGATGTGCAAACGCTCCAAACAGAGCTTCAGCAATCCCAAAAATGGCAGGACAGGACATGGGATGT


TGTGAAGTGGGTAGGCGGAATCTCAGCGGGCCTAGCGGTGAGCGCTTCCATTGCCCTGTTCGGGTT


GGTCTTTAGATTTTCTGTTTCCCTGCCATAAAAGCACATTCTTATAAGTCATACTTGTTTACATCA


AGGAACAAAAACGGCATTGTGCCTTGCAAGGCACAATGTCTTTCTCTTATGCACAGATGGGGACTG


GAAACCACACGCACAATTCCCTTAAAAAGCAACCGCAAAAAATAACCATCAAAATAAAACTGGACA


AATTCTCATGTGggccggccaaaatgaagtgaagttcctatactttctagagaataggaacttcta


tagtgagtcgaataagggcgacacaaaatttattctaaatgcataataaatactgataacatctta


tagtttgtattatattttgtattatcgttgacatgtataattttgatatcaaaaactgattttccc


tttattattttcgagatttattttcttaattctctttaacaaactagaaatattgtatatacaaaa


aatcataaataatagatgaatagtttaattataggtgttcatcaatcgaaaaagcaacgtatctta


tttaaagtgcgttgcttttttctcatttataaggttaaataattctcatatatcaagcaaagtgac


aggcgcccttaaatattctgacaaatgctctttccctaaactccccccataaaaaaacccgccgaa


gcgggtttttacgttatttgcggattaacgattactcgttatcagaaccgcccagggggcccgagc


ttaagactggccgtcgttttacaacacagaaagagtttgtagaaacgcaaaaaggccatccgtcag


gggccttctgcttagtttgatgcctggcagttccctactctcgccttccgcttcctcgctcactga


ctcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt


atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaa


ccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa


tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctgg


aagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctccc


ttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcg


ctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaacta


tcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggat


tagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgggctaactacggctacac


tagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtag


ctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac


gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaa


cgacgcgcgcgtaactcacgttaagggattttggtcatgagcttgcgccgtcccgtcaagtcagcg


taatgctctgcttttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgtt


catccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggcc


ccagcgctgcgatgataccgcgagaaccacgctcaccggctccggatttatcagcaataaaccagc


cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaatt


gttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccatcgcta


caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaa


ggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttg


tcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactg


tcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagt


gtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaa


ctttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgt


tgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacca


gcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacgga


aatgttgaatactcatattcttcctttttcaatattattgaagcatttatcagggttattgtctca


tgagcggatacatatttgaatgtatttagaaaaataaacaaataggggtcagtgttacaaccaatt


aaccaattctgaacattatcgcgagcccatttatacctgaatatggctcataacaccccttgtttg


cctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagc


gccgatggtagtgtggggactccccatgcgagagtagggaactgccaggcatcaaataaaacgaaa


ggctcagtcgaaagactgggcctttcgcccgggctaattatggggtgtcgcccttattcgactcta


tagtgaagttcctattctctagaaagtataggaacttctgaagtggggcctgcagg (SEQ ID NO: 17)


 


SEQ ID NO: 18


plasmid pJB825t








1st underlined sequence
Upstream homology region for TS1 chromosomal 



integration site


1st italic sequence
cI promoter with JCC138 rbcL ribosome-binding 



sequence


1st bold sequence
tll1313 (adm) coding sequence


1st lower case sequence
Native intergenic sequence


2nd bold sequence
tll1312 (aar) coding sequence


2nd italic sequence
kanHTK coding sequence; thermostable kanamycin 



selection marker


2nd underlined sequence
Downstream homology region for TS1 chromosomal 



integration site


2nd lower case sequence
Vector backbone







TGGGAGTCAATAAACCCGATGTGCGTTGGATTTGCCACTACCAGCCGCCCCTGCAACTCAGTGAA


TATCTCCAAGAGGTGGGACGCGCTGGGCGAGATGGCGAAGCGGCACAGGCCCTGGTTTTGGTGAG


CGATCGCTGGGGCTTGGATCGCGAAGATCAACAGCGTTGGTCTTTTTTTCAGCACCAAAGTCAAG


ACACCTACAATCGCGCCATGGCACTTCAGACGCAGCTGCCCCTCCAGGGTAATCTGCAGCAACTG


CGGCAACACTTTCCTGAAGTGGAATTGACCCTGGCATTACTGCATCAACAGGGGGCCCTCCGCTG


GCAAGATCCCTTTCACTATTGCCGTCAACCCTTGGCACAGGTGCCACCCCCACCCAAAGACCCTC


AAGAACAGTTGATGCAAAAGTTCCTCTATCACCGGGGCTGCCGCTGGCAGTTTCTCCTCCAAGCC


TTTGGTTTTGCCACTGAGGCAAGGGGATTCCACTGTGGCCATTGCGATCGCTGTCGGCCGCCGCA


CCGCTCCCGCAAAATACCGTAAATTGCCAGCGCTGTATCACTGGAATATTGGGTACACTGGCACA


TAGAACGGTCGCTTTACCATTGGTAGGCAAAAGTTTCTCAGCAGTCATTCTGTTGCCGCAAGGTA


GGGGTTGCAGGCATGGGGCTACTACAAGTTGAGGAAATTCGCGAAGCACTTCAAGATGTGCTTTC


AGAACACGCCCTTGTTGTGCAAGTTAATCAGTTTCGCAACCAATTAAACATTATTTTGAACAAGC


CCCCCGGCACCGTTGCCCATTATTCTGCCCTAGCGGATTTTCTCAAGTCGCGCTTGGGACAGTTT


CATCTCAATGATATTGACCGCATTAAAATAATTGGCCGCATACAGGGTTCGCCTAAACCCGATTG


GGAAGAGGTCATTGATCTACGTCCCCCCAACCCAGCCCTAGCTGCCCCTGTGTATGCTTCTTCTG


CCCCGTGGGTGGTGGCGATCGCTGCTGGCTTTGTCAGTTTACTGGTGATCTTTAGCTATCACCTT


GGTCAGTAGCAGCAACAGCAACGGCTGTAGCCGTTGATCGAAGGTTCCTTTGGTCAAAAGGGCGT


CGTGATGACGGACTTTAAGTGGCACATTGAGGGTGGTACAGGGTTTATTGTCGGGGTTCTTAAAA


ACTACAGTAAAGGGTATTTTCGCTTAGTTCAGGCGGACTTTGAACTCTTTGACCAAGGCGGTCAG


CAAGTTGGGACAGTGGCGGTACAGGTTTATGGTCTTGGCCCTGAGGAAACATGGCAATTCCGTGA


ACTGATAGCCAATCATCAGGCAGTGCGAGCACGGCTGGTAAAATTACAGTCATTCAATTAAGGTT


TTTCTAATGTTTAGGTTTCCCCAGCAGGGAGCGACACCGCTTGCTATGGCACACCTTAAAGCCCT


GATCTTTGATGTCGATGGCACCTTAGCAGATACGGAGCGGGATGGCCATCGTATCGCCTTCAACA


AGGCCTTTGCCGCCGCTGGTCTAGATTGGGAATGGGACATTCCCCTCTATGGTCAACTCCTGGCG


GTGGCTGGGGGCAAGGAGCGGATCCGGTATTACCTTGAGTGCTTTCGTCCCGATTGGCCACGTCC


CCAAAATTTGGATGCTCTGATTGCCGATTTACACAAGGCCAAGACCCGCTATTATACCGAGCTAT


TGGCGGCAGGGGCTATTCCCCTGCGGCCGGGGGTGAAACGGCTCCTCACTGAAGCCCGGGAAGCA


GGATTACGTTTGGCGATCGCCACCACGACCACCCCTGCCAATGTCACCGCACTCCTTGAAAATGC


CCTCGCTCCTGATGGCGTCAGTTGGTTTGAGATAATTGCTGCCGGGGATGTAGTTCCAGCCAAGA


AACCCGCGCCCGACATTTACTTCTACACGCTTGAAAAGATGCGCCTCTCACCCCAAGAGTGCCTT


GCCTTTGAGGATTCCGCCAATGGGATTCAGGCGGCCACTGCCAGTCACCTAGCGACCATTATCAC


GATTACCGACTACACCAAGGATCATGATTTTCGTGATGCAGCGCTGGTCTTGGATTGCTTAGGGG


AACCGGACTACCCCTTTCAGGTTCTGCGCGGTGAGGTGGGTTGGACAACCTATGTGGATGTCCCC


CTATTGCGATCGCTGCACCAGCAGTGGACAAGCACGTTGAGTCAGGGATAATTTTCTGGCCGCAG


CGTTTTACATTGAATATGACCCCCTTAGTCTGAGGATCAAGGAACATAATGTACACGATTGATTT


AATTCTGCGTCATGTCCCCATGCCCGTCAGCATTGAACGCAAGGAAAGTGCAGCAGCGATGGCAG


TCTATCAGCAAATTCAGCAGGCCATGGCCAGTGGTACTCCAACTTTCCTCGAACTGACGTGCGAT


CGCCAAGTGGGCAAGAAGTTAACGGTGCTCACCTCAGAAATTGTCGCCGTGCAAATGGCGGATAA


GGATGCCCCCTCCAGTACTATCAGTCGTGGGGGATTCTTTGCTCAATTAGTGCAGCAAACCAGCA


ACTGAGGGAAAATGCCTCAATAAAGTTGAGTTTTTCTTGGCAATGCTGATTCTTTGCCGTTAGGA


TACTAAGCAGACCGATCCGTAGGGGAACGTGAAGCAAATCCTCCCCGTCTGAAAGTCAGGTATCT


CTGGTGTGTCGTAATAGGGTTGTCTATGGTGCAGCGTTTCCTGCCGGTTCTGATTTTGTTGGGGT


GTAGTTTTGGTCTTGCGACCCCTGCCCTTGTGCGTGCCCAAGCCAATCAGGGCTTTACGTTTACT


TGGGGTGAGGGGCCGAGTGGCCGACAGCAGTTGCAATACCACTTAGATAACGGCACCCCCGGTTT


TATGGGCGATCGCTATTGGCTGCGGCTGGGTCAGCAGAAAGTGGCCATCAATCGCATTAACATTA


CCTATCCCGACTACTACAACGGTATTATTGATCCCAAAGGCATTGAGGTGCGCATCGGTGGCGAT


CGCGGCAATCGCTTCTTCCAATTTCGCCGTGACCCCGGCACCAAAATTCAATTGGCGGAAGTCTC


CGTTGATCGCGATAACCGCGTGATTGATATTGTGCCGGCTGAGGTGATTCCCGCCGGAACACCGG


TGCAAGTTATTCTCAATAATGTGCGCAACCCTAACAATGGCGGCATGTACTATTTCAATGCCCGC


ATTGGCTCCCCTGGAGATATTCCCCTCATGCGCTACGTTGGCACCTGGATTCTCAGCATTGCCAA


TAACTAAAACCCGTCAAACTCGAGCATTGGTGAGCGGGTTAGCCATTTCTAACTATTGCGGGGCG


ATCGCCCTAGACTAGTTTTTTGTCTATTATTGCCGGTTCACTCTTTACACCAGATGCCAGATTCC


GTTAGGTCTTCATTCCCCTCCATTTCTCCTCTGCTCACGCCTCTGATGTACCGCCTCGTGGGGGA


CGTTGTCCTGCGGCGCTATTTTCGTACCCTTGAGGTGCAAGGGCAGGAGCGGGTGCCCCAAAGGG


GTCCAGTGATCTTGGCCCCCACCCACCGTTCCCGCTGGGATGCGCTGATTATTCCCTATGTCACT


GGGCGGCGGGTGAGTGGGCGCGACCTCTACTACATGGTGTCCCACGATGAGATGTTGGGACTACA


GGGCTGGGTGATTGCTCAGTGTGGCGGTTTTCCCGTCAATACCCAAGCGCCTTCGGTGAGTGCGT


TGCGTACGGGTGTGGAACTGCTCCGGCAGGGGCAAGCCTTGGTGGTGTTCCCTGAGGGGAATATC


TTTCGCGATCGCCAGATTCATCCCCTCAAGCCGGGGTTGGCTCGCTTAGCCCTTCAGGCGGCCCA


GCGCTGTGAACAAGCAATCCAGATTCTGCCAATTTTACTCGATTATGCCCAGCCCTACCCACAGT


GGGGAAGTGCGGTCAAGGTAATCATTGGGGCTCCCTTGAGTACCGACAATTACGATGCCAGCCGG


CCAAAAAGTGCTGCCCAACAACTGACCAGTGATCTCTTTAGAAGACTTCAGCAGCTCCAAGGGGG


GCGATCGCCCCTGTGTTTTGCTTAGACCTCAAACTTCCATCCCCGCGGCCGCTCTTGATAACCCA


AGAGGGCATTTTTTAGGCGCGCCTCGAGTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGT


GATAATGGTTGCAGGATCCTTTTGCTGGAGGAAGAATTCATGACAACGGCTACCGCTACACCTGT


TTTGGACTACCATAGCGATCGCTACAAGGATGCCTACAGCCGCATTAACGCCATTGTCATTGAAG


GTGAACAGGAAGCTCACGATAACTATATCGATTTAGCCAAGCTGCTGCCACAACACCAAGAGGAA


CTCACCCGCCTTGCCAAGATGGAAGCTCGCCACAAAAAGGGGTTTGAGGCCTGTGGTCGCAACCT


GAGCGTAACGCCAGATATGGAATTTGCCAAAGCCTTCTTTGAAAAACTGCGCGCTAACTTTCAGA


GGGCTCTGGCGGAGGGAAAAACTGCGACTTGTCTTCTGATTCAAGCTTTGATCATCGAATCCTTT


GCGATCGCGGCCTACAACATCTACATCCCAATGGCGGATCCTTTCGCCCGTAAAATTACTGAGAG


TGTTGTTAAGGACGAATACAGCCACCTCAACTTTGGCGAAATCTGGCTCAAGGAACACTTTGAAA


GCGTCAAAGGAGAGCTCGAAGAAGCCAATCGCGCCAATTTACCCTTGGTCTGGAAAATGCTCAAC


CAAGTGGAAGCAGATGCCAAAGTGCTCGGCATGGAAAAAGATGCCCTTGTGGAAGACTTCATGAT


TCAGTACAGTGGTGCCCTAGAAAATATCGGCTTTACCACCCGCGAAATTATGAAGATGTCAGTTT


ATGGCCTCACTGGGGCATAAtggtggcttaacgtatcgttacatttcagtcaccacacgcttgtA


TGTTTGGATTAATTGGTCATCTGACGAGTCTGGAGCACGCCCAAGCCGTTGCCCATCAGTTGGGT


TACCCCGAATATGCCGATCAAGGCTTGGAATTTTGGTGTATGGCACCGCCGCAGATCGTCGATGA


GATTACGGTGACGAGCGTAACGGGCAAAACTATCTATGGCAAATACGTTGAGTCCTGCTTTTTAC


CAGAGATGCTGGCCAACCAGCGGGTGAAGGCAGCGACTCGCAAAGTTATTAACGCCATGGCCCAT


GCCCAAAAGCACAACATTGACATTACGGCCTTGGGGGGCTTCTCCTCGATCATCTTTGAGAACTT


TGATCTGGAGAAAATGTCCCACATTCGCAACATTGAACTGGACTTTCGCCGCTTTACAACGGGGA


ATACCCATACCGCCTATATCATCTGCCAACAAATTGAGCAGGCGGCGCCCCAAGTGGGGATTGAT


TTGCGGCAGGCAACCGTGGCTGTTTGTGGGGCTACGGGGGATATTGGTAGTGCCGTCTGCCGTTG


GTTGAATACCTGTTTAGATGTGCAAGATCTCTTACTCGTAGCACGGAATCGCGATCGCCTGCTGG


AGCTACAGGCGGAATTGGGACGGGGGAAAATCCTCGACTTGATGGAGGCGCTGCCCCTTGCCGAT


ATTGTGGTTTGGGTGGCCAGTATGCCCAAGGGAGTTGAGCTGAGCATTGAGCAGTTAAAACGCCC


CTCCCTGATGATTGATGGTGGTTATCCCAAAAATATGGCCACCAAAATTCAGCACCCCCAGATTC


ATGTTCTCAATGGTGGCATTGTCGAGCATGCCCTCGACATTGACTGGAAAATTATGGAAATTGTG


AATATGGATGTGCCCTCGCGGCAGATGTTTGCCTGTTTTGCAGAGGCTATGCTTTTAGAGTTCGA


GGGCTGGCACACCAATTTCTCTTGGGGACGCAATCAAATCACTGTGGAAAAGATGCAGCAAATTG


GTGAGGTCTCCCGTAAACATGGATTTCAGCCACTACTGTTGAATCCTCAGTAAGCGGCCGCAAAA


AAAACGGGCCGGCGTATTATCGCCGGCCCGAGTAACACCGTGCGTGTTGACTATTTTACCTCTGG


CGGTGATAATGGTTGCAGGATCCTTTTGCTGGAGGAAAACCATATGAAAGGACCAATAATAATGA


CTAGAGAAGAAAGAATGAAGATTGTTCATGAAATTAAGGAACGAATATTGGATAAATATGGGGAT


GATGTTAAGGCAATTGGTGTTTATGGCTCTCTTGGTCGTCAGACTGATGGGCCCTATTCGGATAT


TGAGATGATGTGTGTTCTGTCAACAGAGGGAGTAGAGTTCAGCTATGAATGGACAACCGGTGAGT


GGAAGGCGGAAGTGAATTTTTATAGCGAAGAGATTCTACTAGATTATGCATCTCGGGTGGAACCG


GATTGGCCGCTTACACATGGTCGATTTTTCTCTATTTTGCCGATTTATGATCCAGGTGGATACTT


TGAGAAAGTGTACCAAACTGCTAAATCGGTAGAAGCCCAAAAGTTCCACGATGCGATCTGTGCCC


TTATCGTAGAAGAGCTGTTTGAATATGCAGGCAAATGGCGTAATATTCGTGTGCAAGGACCGACA


ACATTTCTACCATCCTTGACTGTACAGGTGGCAATGGCAGGTGCCATGTTGATTGGTCTGCATCA


TCGCATCTGTTATACGACGAGCGCTTCGGTCTTAACTGAAGCAGTTAAGCAACCAGATCTTCCTC


CAGGTTATGTCCAACTGTGCCAGCTCGTAATGTCTGGTCAACTTTCCGACCCTGAGAAACTTCTG


GAATCGCTAGAGAATTTCTGGAATGGGGTTCAGGAGTGGGCGGAACGACACGGATATATAGTGGA


TGTGTCAAAACGCATACCATTTTGATGTCTAACCCCCTTCCTTGCCCACAGCTTCGTCGATGGCG


CGAAATTTCGGGTAAATATAATGACCCTCTTGATAACCCAAGAGGGCATTTTTTAGGCGCGCCCT


AAGCGTCCGTAGGCACAATTAAGGCTTCAAATTGTTGGCGAAGCTGCTCAGTCACTTCCTTGACG


GCTTGCCGTGCCCCTTGGCGATCGCGCCGGTACAGAGGCCAATAGCTCTCTAAATTGAGAGGGTC


GCCGACACTGAGGCGCACCTGCCGCAAACCCACCAAACGATTGAGATTCGAGCTTTTTCCCTCTA


GCCAATCAAATGTGCGCCAGAGAATCAGCGCGACATCTGCAAAGCGATGAATCGTGAATTTCTCA


CGGATATAGCTACCCGTAATTGAGGTAAATCGCTCCGCAAGACGCATATGACGCAATCGCACATT


GGCTTCCTCGGCCAACCAATCGGCTAGGCAGCGCTCTACGGCCGAAAGTTGTGCCAAATCACTGC


GAAACATCCGTTCCCAAGCAGCCTGTTCAATGCGTCGGCAGCGACTCACAAAATCGGCACTGGGC


TTCAGACCAAAGTAGGACTCTGCCACCACAAGGGCGCTGTTGAGGAGGCGCTGAATTCGCGCTGC


CAATTTAGCATTGGCAGAGTCAAAGGGGGGCAGTTCGGGAAAATCTTGACCATAGGAGGTGGCAT


AAAAAGCCTCCAGGCGATCCAAGAGGTGGATCGCTAAATTCAGCAGGCGGCGGTAGAGGTCGTCT


GGCTGGGTACTGTGAGAATCTGTAGGGCACCCAAGGCGGTTCTCCAGTTGTGCCATCAGCCTTGC


CATGCGCTCCCAAGAGGGCTGACTGAGGCTGTACTGAATGCCAATGGGAAGAATGACCACGGGGA


GCGATCGCCCCGCCTTGGCTAAATCTTCTAGACACCAAAATCCCAGTTGGGCCACCCCCGGCTCC


AAAGGTGCGACCAGTTCGTTGTGCTCATTCGTTGCTCCCTCCGGCGCTGCCGCTAGGGGAAATCG


TCCTCCGAGAAGTAGCTCCCGCGCTGAGCGCAGGGCTTGGCTATCGAGCTTACCGCGCATGATGG


AAATCCCCCCCAACCGTGAAAAGAGCCAACCAATCTGCGCCCCTGCCCAGAGGGGAATCCCGCGA


TCGTAGAGAAAATAGCCATTTGTCGGCGGACGCAAGGGAATGCCCAGCCGCCGTGCTGTTTGCGG


CAGTAAATGCCACATCAAATAGCCCATCACCAACGGATCATCCGTACAGGGATGGCGAAAGGCAA


TGAGGAGCCGGACCTGTCCCTGCTGAAACTGCTGGTAATAACGGGCAAGGGTCTCCACATTCACC


CCTTCAACCCGCTGTAGCCCAAGACCATAGCGAATGTAGAGGGGCAGGAGTCTTGCTACTGTCCA


CCAGACGGGGTAGCTAAACCGCTGGGGGAGAAAATGCAACGGCGGTTGGGCAGTTGTCACTACAC


TGGACATTAGGCAAGCTCCTCAGGGCAATGGCTAAACTGAGGCAGTGGCCAACTCCGCAATTAAC


TGCTCTAACATCGGTTGATCGGCCCAATAGACAGCATTACAAAACTGACAGGTGGCTTCTGCCTT


TGCCTCTGTGGCTAGGATATCTCTTAATTCTGCCTCCCCTAGGAGCTTGAGTGCCGCTAACATCC


GTTCATGGGAACAGCCACAGTGGAAGCGCACCATTTGCCGTTGGGGCAAGATTTGTAAATCCATA


TCCCCTAAGAGTTCCTGAAAGATATCTGGCAGTGTCCGCCCTGCCTGTAGCAGTGGTGTAAAGCC


CTTAAGATTGGCCACCCGTTGTTCAAGGGTCGCGATCAGGTGTTCATCATTGGCCGCTTTGGGTA


GCACCTGTAACATCAACCCACCGGCGGCAGTCACCCCGGACTCTTCGACAAAAACACCCAACATC


AGGGCGGAGGGGGTTTGCTCTGAGGTGGCGAGGTAGTAGGTGATGTCTTCTGCAATTTCGCCGGA


GACTAGCTCCACCGTGCTGGAATAGGGGTAGCCGTAGCCAAGATCGTGGATGACGTAGAGATATC


CCTGATGGCCCACCGCTGCCCCCACATCGAGTTTGCCCTTGGCATTGGGGGGCAGTTCAACACTG


GGGTACTGCACATAGCCGCGAACTGTGCCATCGGCACCAGCATCGGCAAAAATGGTTCCTAGGGG


ACCGTTGCCCTGAATGCGCACATTCACCCGTGCTTGGGGCTGTTTGAAACTGGAGGCAAGGATTA


AGCCTGCGGCCATGGTTCGTCCCAAGGCCGCTGTGGCCACGTAGGACAGTTGGTGACGTTTGCGG


GCTTCATCAGTGAGTTGAGTGGTAATCACACCTACGGCCCGGATGCCTTCGGCAGCGGCAGTTGC


TCGCAACAGAAAATCGGCCATGTTCAACCTACGAAATGTTTTGTTACATTTAGTGTGACATACTC


CCACCGCTGACCAGGGCACAATGGGGCAAAAAACCATCAATCCTGCCTTTGGTGACCGATCCAGT


ACAGCCAGCCAGGGCTTAAGACTGGGAAGACCCCTAGCACTGGGGCTAGAAAATTGGCGATGATA


GGCAAGCAATAGTCATTCAGCGTCCAGTCATTCCGCCTATGGCCATGCCCCTCACTGTCTTGCCT


GCCACAACTGTTTTGACAGAAGCGACTCAATTGCCCCAGGGCGGCTTGATTACGGAGATTCCGAC


GCTGGCGATCGCCCACCGTTTGGCCCAGCAGTTGCGCCGCCATTGGCCCCTAGAGACCCCCTTAA


CGCTGATTGATGCGCAATACCAGAGTATCCCCCTGACCCTTGGGGAATTGGCCGAGCTCACCGAT


GCCAACTGTCCTTTACAGCTCTATGTGCCGCCCCCCTTGCCAGAGGCCTTGACGCAATTTCAACG


CCTGATGGATGTGGTTCGAGAGCTGCGCCATCCGGAGCGTGGCTGTCCTTGGGATTTGCAGCAAA


CCCCAACCAGTCTCATTCCCTATGTCCTTGAGGAAGCCTATGAAGTGGTACATGCCCTGCAGGAG


GGAGATGCGGGGGCGATCGCCGAAGAATTGGGAGACCTGTTGCTTCAAGTTGTTCTCCAGAGCCA


ACTTGCCCAAGAAGCCGGCCAATTTACCCTTGCTCAAGTCATTCAAAGGATTACCGATAAACTCA


TCCGCCGCCATCCCCACGTCTTTGGTGAAGTGGCACTCACCACTGCTCAAGAGGTGCGCGACCAA


TGGGAGCAAATCAAAGCGGCTGAAAAAGGCACCGAACTCCCCCTGAGTCAAACGCTGCAACGTTA


CGCACGCACCCTCCCACCCCTGATGGCCGGCATGAAAATTGGTGAGCGAGCCAGTCGCGCTGGCC


TCGATTGGCCGACGATTAGTGGTGCATGGGAGAAATTTTACGAGGAACTGGCGGAGTTTCAGGAG


GCCCTTCTGCAAGGGAATGCTGAGCAACAGGCAGCGGAATTAGGAGACCTGCTCTTCAGTGTGAT


TAACCTTGCCCGCTGGTGCCAACTGGATCCTGTTAATGCCCTGCAACAAACCTACCAACGCTTTA


TTCAACGCTTGGCCTGTATTGAGGCAGTCATCGATCGCCCCCTTGAGACGTACACCCTAGAAGAA


CTAGAAGCCCTCTGGCAACAGGCCAAAGTACAGTTAGCCACCGACAGCGAGGCAACCCCTATGGA


GACTGAGGAAGAGGCCTAGTCCGCTGCGGCCCTTGCCACCTTCAGTTCATCGAGATTCCACAGGG


GGCCCCCCAGCGCCGTGGGCTTGGCGCCAATGACATGATTGCGAAAAGCTGTAAGGGAGAGGGGA


TTCACGAGGTAAATAAAGGGGAGATATTCCTGAGCTAGTCGTTGGGCTTCCGCATAAATTTGCTG


CCGTCGTTCCAGATTGAGCTCCTGGGCACCTTGGACATACAGGTCACTGATGCGCTGCTCCCAGT


CAGCGACGACTCGACCCGTAATGGGTGGTTGATTCGGTGACGGTTGCTGATTGAATGTATGCAAA


AGGCCATCCACACGCCAGATATTGGCACCGCTATTGGGTTCATTGCCCCCCCCAGTAAAGCCGAG


GATATGGGCTTCCCACTCTAGGGAATTGGAGAGACGATCCACGAGGGTACCAAAGGCCAAAAATT


GCAGATCCACCTGCATGCCGATCGCCCCTAGGTCCTGCTGAACTTGCGTCGggccggccaaaatg


aagtgaagttcctatactttctagagaataggaacttctatagtgagtcgaataagggcgacaca


aaatttattctaaatgcataataaatactgataacatcttatagtttgtattatattttgtatta


tcgttgacatgtataattttgatatcaaaaactgattttccctttattattttcgagatttattt


tcttaattctctttaacaaactagaaatattgtatatacaaaaaatcataaataatagatgaata


gtttaattataggtgttcatcaatcgaaaaagcaacgtatcttatttaaagtgcgttgctttttt


ctcatttataaggttaaataattctcatatatcaagcaaagtgacaggcgcccttaaatattctg


acaaatgctctttccctaaactccccccataaaaaaacccgccgaagcgggtttttacgttattt


gcggattaacgattactcgttatcagaaccgcccagggggcccgagcttaagactggccgtcgtt


ttacaacacagaaagagtttgtagaaacgcaaaaaggccatccgtcaggggccttctgcttagtt


tgatgcctggcagttccctactctcgccttccgcttcctcgctcactgactcgctgcgctcggtc


gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcagg


ggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccg


cgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagt


cagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgt


gcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcg


tggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg


ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttga


gtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagag


cgaggtatgtaggcggtgctacagagttcttgaagtggtgggctaactacggctacactagaaga


acagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg


atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgca


gaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgac


gcgcgcgtaactcacgttaagggattttggtcatgagcttgcgccgtcccgtcaagtcagcgtaa


tgctctgcttttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttca


tccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccc


cagcgctgcgatgataccgcgagaaccacgctcaccggctccggatttatcagcaataaaccagc


cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaat


tgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccatcgc


tacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgat


caaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatc


gttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct


tactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgag


aatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacat


agcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatctt


accgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctttta


ctttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagg


gcgacacggaaatgttgaatactcatattcttcctttttcaatattattgaagcatttatcaggg


ttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggtcagtg


ttacaaccaattaaccaattctgaacattatcgcgagcccatttatacctgaatatggctcataa


caccccttgtttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaag


tgaaacgccgtagcgccgatggtagtgtggggactccccatgcgagagtagggaactgccaggca


tcaaataaaacgaaaggctcagtcgaaagactgggcctttcgcccgggctaattatggggtgtcg


cccttattcgactctatagtgaagttcctattctctagaaagtataggaacttctgaagtggggc


ctgcagg (SEQ ID NO: 18)


 


SEQ ID NO: 19


cI promoter used for transcription in pJB886. Terminal NotI and NdeI 


sites used for promoter exchanges are capitalized.


GCGGCCGCtcgagtaacaccgtgcgtgttgactattttacctctggcggtgataatggt


tgcaggatccttttgctggaggaaaacCATATG (SEQ ID NO: 19)


 


SEQ ID NO: 20


cpcB promoter used for transcription in pJB887.


GCGGCCGCttcgttataaaataaacttaacaaatctatacccacctgtagagaagagtc


cctgaatatcaaaatggtgggataaaaagctcaaaaaggaaagtaggctgtggttccct


aggcaacagtcttccctaccccactggaaactaaaaaaacgagaaaagttcgcaccgaa


catcaattgcataattttagccctaaaacataagctgaacgaaactggttgtcttccct


tcccaatccaggacaatctgagaatcccctgcaacattacttaacaaaaaagcaggaat


aaaattaacaagatgtaacagacataagtcccatcaccgttgtataaagttaactgtgg


gattgcaaaagcattcaagcctaggcgctgagctgtttgagcatcccggtggcccttgt


cgctgcctccgtgtttctccctggatttatttaggtaatatctctcataaatccccggg


tagttaacgaaagttaatggagatcagtaacaataactctagggtcattactttggact


ccctcagtttatccgggggaattgtgtttaagaaaatcccaactcataaagtcaagtag


gagattaatCATATG (SEQ ID NO: 20)


 


SEQ ID NO: 21


lacI-trc promoter used for transcription in pJB889. Terminal NotI and 


NdeI sites used for promoter exchanges are capitalized.


GCGGCCGCgaaggcgaagcggcatgcatttacgttgacaccatcgaatggtgcaaaacc


tttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatatgaa


accagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttccc


gcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcg


atggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtc


gttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcg


cggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaa


cgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcag


tgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcct


gcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtatt


attttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtca


ccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctgg


ctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggc


gactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgt


tcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccatta


ccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaa


gacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggg


gcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatc


agctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaacc


gcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgact


ggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattgatctggttt


gacagcttatcatcgagctcgactgcacggtgcaccaatgcttctggcgtcaggcagcc


atcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctca


aggcgcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaa


tattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtg


agcggataacaatttcacacaggaaacagCATATG (SEQ ID NO: 21)


 


SEQ ID NO: 22


EM7 promoter used for transcription in pJB888. Terminal NotI and NdeI 


sites used for promoter exchanges are capitalized.


GCGGCCGCtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaa


ggtgaggaactaaCATATG (SEQ ID NO: 22)


 


SEQ ID NO: 23


DNA sequence of pJB1279.








1st underlined sequence
Upstream homology region for SYNPCC7002_A0358


1st italic sequence
P(nir07) promoter; this promoter is a synthetic 



construct based on the nirA promoter from 



Synechococcus sp. PCC 7942. (Shin-Ichi Maeda et 



al. (1998) J. Bacteriol., 180: 4080-4088.


2nd italic sequence
aadA coding sequence; spectinomycin selection 



marker


2nd underlined sequence
Downstream homology region for SYNPCC7002_A0358


Lower case sequence
E. coli vector backbone (pUC ori, kan)







ACAACTCGGCTTCCGAGCTTGGCTCCACCATGGTTATATCTGGAGTAACCAGAATTTCG


ACAACTTCGACGACTATCTCGGTGCTTTTACCTCCAACCAACGCAAAAACATTAAGCGC


GAACGCAAAGCCGTTGACAAAGCAGGTTTATCCCTCAAGATGATGACCGGGGACGAAAT


TCCCGCCCATTACTTCCCACTCATTTATCGTTTCTATAGCAGCACCTGCGACAAATTTT


TTTGGGGGAGTAAATATCTCCGGAAACCCTTTTTTGAAACCCTAGAATCTACCTATCGC


CATCGCGTTGTTCTGGCCGCCGCTTACACGCCAGAAGATGACAAACATCCCGTCGGTTT


ATCTTTTTGTATCCGTAAAGATGATTATCTTTATGGTCGTTATTGGGGGGCCTTTGATG


AATATGACTGTCTCCATTTTGAAGCCTGCTATTACAAACCGATCCAATGGGCAATCGAG


CAGGGAATTACGATGTACGATCCGGGCGCTGGCGGAAAACATAAGCGACGACGTGGTTT


CCCGGCAACCCCAAACTATAGCCTCCACCGTTTTTATCAACCCCGCATGGGCCAAGTTT


TAGACGCTTATATTGATGAAATTAATGCCATGGAGCAACAGGAAATTGAAGCGATCAAT


GCGGATATTCCCTTTAAACGGCAGGAAGTTCAATTGAAAATTTCCTAGCTTCACTAGCC


AAAAGCGCGATCGCCCACCGACCATCCTCCCTTGGGGGAGATGCGGCCGCTTGTAGCAA


TTGCTACTAAAAACTGCGATCGCTGCTGAAATGAGCTGGAATTTTGTCCCTCTCAGCTC


AAAAAGTATCAATGATTACTTAATGTTTGTTCTGCGCAAACTTCTTGCAGAACATGCAT


GATTTACAAAAAGTTGTAGTTTCTGTTACCAATTGCGAATCGAGAACTGCCTAATCTGC


CGAGTATGCGATCCTTTAGCAGGAGGAAAACCATATGAGATCTGTAGTAGGATCCCTCG


AGAGTGAGAGCCGGCGAGCTCATAGTATGTACATGATGACTGTACCCATGGTTGAATTC


GGTTTTCCGTCCTGTCTTGATTTTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGC


ATTTGTTTTTGTTTATTGCAAAAACAAAAAATATTGTTACAAATTTTTACAGGCTATTA


AGCCTACCGTCATAAATAATTTGCCATTTACTAGTTTTTAATTAACCAGAACCTTGACC


GAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTT


TGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGAT


GTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAG


TTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGT


TGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCG


CAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTA


AGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTC


CCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACA


TCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAAT


GACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCT


GACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTG


ATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAAC


TCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTG


GTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGG


AGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGA


CAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGT


GAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGAC


GCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCAC


AGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTA


CACAAATTGGGAGATATATCATGAGGCGCGCCTGATCAGTTGGTGCTGCATTAGCTAAG


AAGGTCAGGAGATATTATTCGACATCTAGCTGACGGCCATTGCGATCATAAACGAGGAT


ATCCCACTGGCCATTTTCAGCGGCTTCAAAGGCAATTTTAGACCCATCAGCACTAATGG


TTGGATTACGCACTTCTTGGTTTAAGTTATCGGTTAAATTCCGCTTTTGTTCAAACTCG


CGATCATAGAGATAAATATCAGATTCGCCGCGACGATTGACCGCAAAGACAATGTAGCG


ACCATCTTCAGAAACGGCAGGATGGGAGGCAATTTCATTTAGGGTATTGAGGCCCGGTA


ACAGAATCGTTTGCCTGGTGCTGGTATCAAATAGATAGATATCCTGGGAACCATTGCGG


TCTGAGGCAAAAACGAGGTAGGGTTCGGCGATCGCCGGGTCAAATTCGAGGGCCCGACT


ATTTAAACTGCGGCCACCGGGATCAACGGGAAAATTGACAATGCGCGGATAACCAACGC


AGCTCTGGAGCAGCAAACCGAGGCTACCGAGGAAAAAACTGCGTAGAAAAGAAACATAG


CGCATAGGTCAAAGGGAAATCAAAGGGCGGGCGATCGCCAATTTTTCTATAATATTGTC


CTAACAGCACACTAAAACAGAGCCATGCTAGCAAAAATTTGGAGTGCCACCATTGTCGG


GGTCGATGCCCTCAGGGTCGGGGTGGAAGTGGATATTTCCGGCGGCTTACCGAAAATGA


TGGTGGTCGGACTGCggccggccaaaatgaagtgaagttcctatactttctagagaata


ggaacttctatagtgagtcgaataagggcgacacaaaatttattctaaatgcataataa


atactgataacatcttatagtttgtattatattttgtattatcgttgacatgtataatt


ttgatatcaaaaactgattttccctttattattttcgagatttattttcttaattctct


ttaacaaactagaaatattgtatatacaaaaaatcataaataatagatgaatagtttaa


ttataggtgttcatcaatcgaaaaagcaacgtatcttatttaaagtgcgttgctttttt


ctcatttataaggttaaataattctcatatatcaagcaaagtgacaggcgcccttaaat


attctgacaaatgctctttccctaaactccccccataaaaaaacccgccgaagcgggtt


tttacgttatttgcggattaacgattactcgttatcagaaccgcccagggggcccgagc


ttaagactggccgtcgttttacaacacagaaagagtttgtagaaacgcaaaaaggccat


ccgtcaggggccttctgcttagtttgatgcctggcagttccctactctcgccttccgct


tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca


ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt


gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttc


cataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcg


aaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgct


ctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc


gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctc


caagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggta


actatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccact


ggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtg


ggctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccag


ttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagc


ggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga


tcctttgatcttttctacggggtctgacgctcagtggaacgacgcgcgcgtaactcacg


ttaagggattttggtcatgagcttgcgccgtcccgtcaagtcagcgtaatgctctgctt


ttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaa


taccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttc


cataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaataca


acctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtga


cgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaaca


ggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcg


tgattgcgcctgagcgaggcgaaatacgcgatcgctgttaaaaggacaattacaaacag


gaatcgagtgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaa


tcaggatattcttctaatacctggaacgctgtttttccggggatcgcagtggtgagtaa


ccatgcatcatcaggagtacggataaaatgcttgatggtcggaagtggcataaattccg


tcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgcca


tgtttcagaaacaactctggcgcatcgggcttcccatacaagcgatagattgtcgcacc


tgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttgg


aatttaatcgcggcctcgacgtttcccgttgaatatggctcatattcttcctttttcaa


tattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtat


ttagaaaaataaacaaataggggtcagtgttacaaccaattaaccaattctgaacatta


tcgcgagcccatttatacctgaatatggctcataacaccccttgtttgcctggcggcag


tagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccg


atggtagtgtggggactccccatgcgagagtagggaactgccaggcatcaaataaaacg


aaaggctcagtcgaaagactgggcctttcgcccgggctaattagggggtgtcgccctta


ttcgactctatagtgaagttcctattctctagaaagtataggaacttctgaagtggggc


ctgcagg (SEQ ID NO: 23)


 


SEQ ID NO: 24


P(nir07) promoter; this promoter is a synthetic construct based on the 


nirA promoter from Synechococcus sp. PCC 7942. (Shin-Ichi Maeda et al. 


(1998). cis-Acting Sequences Required for NtcB-Dependent, Nitrite-


Responsive Positive Regulation of the Nitrate Assimilation Operon in 


the Cyanobacterium Synechococcus sp. Strain PCC 7942. J. Bacteriol.  


180: 4080-4088


GCTTGTAGCAATTGCTACTAAAAACTGCGATCGCTGCTGAAATGAGCTGGAATTTTGTC


CCTCTCAGCTCAAAAAGTATCAATGATTACTTAATGTTTGTTCTGCGCAAACTTCTTGC


AGAACATGCATGATTTACAAAAAGTTGTAGTTTCTGTTACCAATTGCGAATCGAGAACT


GCCTAATCTGCCGAGTATGCGATCCTTTAGCAGGAGGAAAACCAT (SEQ ID NO: 24)


 


SEQ ID NO: 25


DNA sequence of synthetic operon encoding the Cyanothece sp. ATCC 51142 adm and aar


genes








1st underlined sequence
EcoRI site used to subclone operon into 



pJB286alk_p


Italic sequence
3′ UTR sequence containing ribosome-binding 



sequence


1st bold sequence
cce_0778 (adm) coding sequence


Lower case sequence
Intergenic sequence


2nd bold sequence
cce_1430 (aar) coding sequence


2nd underlined sequence
EcoRI site used to subclone operon into 



pJB286alk_p







GAATTCTATAAGTAGGAGGTAAAAACATGCAAGAACTGGCCCTGAGAAGCGAGCTGGAC


TTCAATAGCGAAACCTATAAAGATGCGTATAGCCGTATTAACGCCATTGTGATCGAAGG


CGAGCAAGAAGCATACCAAAACTACCTGGACATGGCGCAACTGCTGCCGGAGGACGAGG


CTGAGCTGATTCGTTTGAGCAAGATGGAGAACCGTCACAAAAAGGGTTTTCAAGCGTGC


GGCAAGAACCTCAATGTGACTCCGGATATGGATTATGCACAGCAGTTCTTTGCGGAGCT


GCACGGCAATTTTCAGAAGGCTAAAGCCGAGGGTAAGATTGTTACCTGCCTGCTCATCC


AAAGCCTGATCATCGAGGCGTTTGCGATTGCAGCCTACAACATTTACATTCCAGTGGCT


GATCCGTTTGCACGTAAAATCACCGAGGGTGTCGTCAAGGATGAGTATACCCACCTGAA


TTTCGGCGAAGTTTGGTTGAAGGAACATTTTGAAGCAAGCAAGGCGGAGTTGGAGGACG


CCAACAAAGAGAACTTACCGCTGGTCTGGCAGATGTTGAACCAGGTCGAAAAGGATGCC


GAAGTGCTGGGTATGGAGAAAGAGGCTCTGGTGGAGGACTTTATGATTAGCTATGGTGA


GGCACTGAGCAACATCGGCTTTTCTACGAGAGAAATCATGAAGATGAGCGCGTACGGTC


TGCGTGCAGCATAActcgagtataagtaggagataaaaacATGTTCGGCTTGATTGGCC


ACCTGACTAGCCTGGAGCACGCGCACAGCGTGGCGGATGCGTTTGGCTACGGCCCGTAC


GCAACCCAGGGTTTAGACCTGTGGTGTAGCGCACCGCCACAGTTTGTTGAGCACTTTCA


TGTCACGAGCATTACGGGCCAAACGATTGAGGGTAAATACATTGAGAGCGCGTTTTTGC


CGGAGATGTTGATTAAACGTCGTATCAAAGCAGCGATCCGTAAGATTCTGAACGCGATG


GCATTTGCGCAGAAGAACAATTTGAACATTACCGCGCTGGGTGGCTTCAGCAGCATTAT


CTTTGAGGAGTTTAATCTGAAGGAGAATCGTCAGGTTCGCAATGTGAGCTTGGAGTTTG


ACCGCTTCACCACCGGTAACACCCATACTGCTTACATTATCTGCCGTCAAGTCGAACAG


GCGAGCGCGAAACTGGGTATCGACCTGTCCCAAGCGACCGTGGCGATTTGCGGTGCCAC


GGGTGATATTGGCAGCGCAGTTTGTCGCTGGCTGGATCGCAAAACCGACACCCAAGAGC


TGTTCCTGATTGCGCGCAATAAGGAACGCTTGCAACGTCTGCAAGATGAACTGGGTCGC


GGCAAGATCATGGGCCTGGAAGAGGCACTGCCGGAAGCAGACATTATTGTGTGGGTTGC


CTCCATGCCGAAGGGCGTGGAGATTAATGCGGAAACCCTGAAGAAGCCGTGTCTGATCA


TTGACGGTGGCTACCCGAAGAATCTGGACACGAAAATCAAGCATCCGGACGTGCACATT


TTGAAGGGTGGTATTGTAGAGCATTCGTTGGACATTGATTGGAAAATCATGGAAACCGT


GAACATGGACGTTCCGAGCCGTCAAATGTTTGCGTGCTTCGCAGAGGCGATCTTGCTGG


AGTTCGAGCAATGGCACACGAACTTCTCGTGGGGTCGCAATCAAATCACGGTGACGAAG


ATGGAACAGATTGGTGAGGCGAGCGTGAAGCATGGTCTGCAACCGCTGCTGTCCTGGTA


AGAATTC (SEQ ID NO: 25)


 


SEQ ID NO: 26


Cyanothece sp. ATCC 51142 aar optimized nucleotide coding sequence


ATGTTCGGCTTGATTGGCCACCTGACTAGCCTGGAGCACGCGCACAGCGTGGCGGATGC


GTTTGGCTACGGCCCGTACGCAACCCAGGGTTTAGACCTGTGGTGTAGCGCACCGCCAC


AGTTTGTTGAGCACTTTCATGTCACGAGCATTACGGGCCAAACGATTGAGGGTAAATAC


ATTGAGAGCGCGTTTTTGCCGGAGATGTTGATTAAACGTCGTATCAAAGCAGCGATCCG


TAAGATTCTGAACGCGATGGCATTTGCGCAGAAGAACAATTTGAACATTACCGCGCTGG


GTGGCTTCAGCAGCATTATCTTTGAGGAGTTTAATCTGAAGGAGAATCGTCAGGTTCGC


AATGTGAGCTTGGAGTTTGACCGCTTCACCACCGGTAACACCCATACTGCTTACATTAT


CTGCCGTCAAGTCGAACAGGCGAGCGCGAAACTGGGTATCGACCTGTCCCAAGCGACCG


TGGCGATTTGCGGTGCCACGGGTGATATTGGCAGCGCAGTTTGTCGCTGGCTGGATCGC


AAAACCGACACCCAAGAGCTGTTCCTGATTGCGCGCAATAAGGAACGCTTGCAACGTCT


GCAAGATGAACTGGGTCGCGGCAAGATCATGGGCCTGGAAGAGGCACTGCCGGAAGCAG


ACATTATTGTGTGGGTTGCCTCCATGCCGAAGGGCGTGGAGATTAATGCGGAAACCCTG


AAGAAGCCGTGTCTGATCATTGACGGTGGCTACCCGAAGAATCTGGACACGAAAATCAA


GCATCCGGACGTGCACATTTTGAAGGGTGGTATTGTAGAGCATTCGTTGGACATTGATT


GGAAAATCATGGAAACCGTGAACATGGACGTTCCGAGCCGTCAAATGTTTGCGTGCTTC


GCAGAGGCGATCTTGCTGGAGTTCGAGCAATGGCACACGAACTTCTCGTGGGGTCGCAA


TCAAATCACGGTGACGAAGATGGAACAGATTGGTGAGGCGAGCGTGAAGCATGGTCTGC


AACCGCTGCTGTCCTGGTAA (SEQ ID NO: 26)


 


SEQ ID NO: 27


Cyanothece sp. ATCC 51142 AAR amino acid sequence


MFGLIGHLTSLEHAHSVADAFGYGPYATQGLDLWCSAPPQFVEHFHVTSITGQTIEGKYIESAF


LPEMLIKRRIKAAIRKILNAMAFAQKNNLNITALGGFSSIIFEEFNLKENRQVRNVSLEFDRFT


TGNTHTAYIICRQVEQASAKLGIDLSQATVAICGATGDIGSAVCRWLDRKTDTQELFLIARNKE


RLQRLQDELGRGKIMGLEEALPEADIIVWVASMPKGVEINAETLKKPCLIIDGGYPKNLDTKIK


HPDVHILKGGIVEHSLDIDWKIMETVNMDVPSRQMFACFAEAILLEFEQWHTNFSWGRNQITVT


KMEQIGEASVKHGLQPLLSW (SEQ ID NO: 27)


 


SEQ ID NO: 28


Cyanothece sp. ATCC 51142 adm optimized nucleotide coding sequence


ATGCAAGAACTGGCCCTGAGAAGCGAGCTGGACTTCAATAGCGAAACCTATAAAGATGC


GTATAGCCGTATTAACGCCATTGTGATCGAAGGCGAGCAAGAAGCATACCAAAACTACC


TGGACATGGCGCAACTGCTGCCGGAGGACGAGGCTGAGCTGATTCGTTTGAGCAAGATG


GAGAACCGTCACAAAAAGGGTTTTCAAGCGTGCGGCAAGAACCTCAATGTGACTCCGGA


TATGGATTATGCACAGCAGTTCTTTGCGGAGCTGCACGGCAATTTTCAGAAGGCTAAAG


CCGAGGGTAAGATTGTTACCTGCCTGCTCATCCAAAGCCTGATCATCGAGGCGTTTGCG


ATTGCAGCCTACAACATTTACATTCCAGTGGCTGATCCGTTTGCACGTAAAATCACCGA


GGGTGTCGTCAAGGATGAGTATACCCACCTGAATTTCGGCGAAGTTTGGTTGAAGGAAC


ATTTTGAAGCAAGCAAGGCGGAGTTGGAGGACGCCAACAAAGAGAACTTACCGCTGGTC


TGGCAGATGTTGAACCAGGTCGAAAAGGATGCCGAAGTGCTGGGTATGGAGAAAGAGGC


TCTGGTGGAGGACTTTATGATTAGCTATGGTGAGGCACTGAGCAACATCGGCTTTTCTA


CGAGAGAAATCATGAAGATGAGCGCGTACGGTCTGCGTGCAGCATAA (SEQ ID NO: 28)


 


SEQ ID NO: 29


Cyanothece sp. ATCC 51142 ADM amino acid sequence


MQELALRSELDFNSETYKDAYSRINAIVIEGEQEAYQNYLDMAQLLPEDEAELIRLSKMENRHK


KGFQACGKNLNVTPDMDYAQQFFAELHGNFQKAKAEGKIVTCLLIQSLIIEAFAIAAYNIYIPV


ADPFARKITEGVVKDEYTHLNFGEVWLKEHFEASKAELEDANKENLPLVWQMLNQVEKDAEVLG


MEKEALVEDFMISYGEALSNIGFSTREIMKMSAYGLRAA (SEQ ID NO: 29)


 


SEQ ID NO: 30


cce_1430 Cyanothece sp. ATCC 51142 coding sequence (aar)


ATGTTTGGTTTAATTGGTCATCTTACAAGTTTAGAACACGCCCACTCCGTTGCTGATGCC


TTTGGCTATGGCCCATACGCCACTCAGGGACTTGATTTGTGGTGTTCTGCTCCACCCCAA


TTCGTCGAGCATTTTCATGTTACTAGCATCACAGGACAAACCATCGAAGGAAAGTATATA


GAATCCGCTTTCTTACCAGAAATGCTGATAAAGCGACGGATTAAAGCAGCAATTCGCAAA


ATACTGAATGCGATGGCCTTTGCTCAGAAAAATAACCTTAACATCACAGCATTAGGGGGC


TTTTCTTCGATTATTTTTGAAGAATTTAATCTCAAAGAGAATAGACAAGTTCGTAATGTC


TCTTTAGAGTTTGATCGCTTCACCACCGGAAACACCCATACTGCTTATATCATTTGTCGT


CAAGTTGAACAGGCATCCGCTAAACTAGGGATTGACTTATCCCAAGCAACGGTTGCTATT


TGCGGGGCAACCGGAGATATTGGCAGTGCAGTGTGTCGTTGGTTAGATAGAAAAACCGAT


ACCCAGGAACTATTCTTAATTGCTCGCAATAAAGAACGATTACAACGACTGCAAGATGAG


TTGGGACGGGGTAAAATTATGGGATTGGAGGAGGCTTTACCCGAAGCAGATATTATCGTT


TGGGTGGCGAGTATGCCCAAAGGAGTGGAAATTAATGCCGAAACTCTCAAAAAACCCTGT


TTAATTATCGATGGTGGTTATCCTAAGAATTTAGACACAAAAATTAAACATCCTGATGTC


CATATCCTGAAAGGGGGAATTGTAGAACATTCTCTAGATATTGACTGGAAGATTATGGAA


ACTGTCAATATGGATGTTCCTTCTCGTCAAATGTTTGCTTGTTTTGCCGAAGCCATTTTA


TTAGAGTTTGAACAATGGCACACTAATTTTTCTTGGGGACGCAATCAAATTACAGTGACT


AAAATGGAACAAATAGGAGAAGCTTCTGTCAAACATGGGTTACAACCGTTGTTGAGTTGG


TAA (SEQ ID NO: 30)


 


SEQ ID NO: 31


cce_0778 Cyanthece sp. ATCC 51142 coding sequence (adm)


ATGCAAGAGCTTGCTTTACGCTCAGAGCTTGATTTTAACAGCGAAACCTATAAAGATGCT


TACAGTCGCATCAATGCTATTGTCATTGAAGGGGAACAAGAAGCCTATCAAAATTATCTT


GATATGGCGCAACTTCTCCCAGAAGACGAGGCTGAGTTAATTCGTCTCTCCAAGATGGAA


AACCGTCACAAAAAAGGCTTTCAAGCCTGTGGCAAGAATTTGAATGTGACCCCAGATATG


GACTACGCTCAACAATTTTTTGCTGAACTTCATGGCAACTTCCAAAAGGCAAAAGCCGAA


GGCAAAATTGTCACTTGCTTATTAATTCAATCTTTGATCATCGAAGCCTTTGCGATCGCC


GCTTATAATATTTATATTCCTGTGGCAGATCCCTTTGCTCGTAAAATCACCGAAGGGGTA


GTTAAGGATGAATATACCCACCTCAATTTTGGGGAAGTCTGGTTAAAAGAGCATTTTGAA


GCCTCTAAAGCAGAATTAGAAGACGCAAATAAAGAAAATTTACCCCTTGTTTGGCAAATG


CTCAACCAAGTTGAAAAAGATGCCGAAGTGTTAGGGATGGAGAAAGAAGCCTTAGTGGAA


GATTTCATGATTAGTTATGGAGAAGCTTTAAGTAATATTGGTTTCTCTACCCGTGAGATC


ATGAAAATGTCTGCTTACGGGCTACGGGCTGCTTAA (SEQ ID NO: 31)

## Claims

1. A method for producing hydrocarbons, comprising:
(i) culturing an engineered cyanobacterium in a culture medium, wherein said engineered cyanobacterium comprises a recombinant acyl-ACP reductase enzyme and a recombinant alkanal decarboxylative monooxygenase enzyme, wherein at least one of said recombinant enzymes is heterologous with respect to said engineered cyanobacterium; and
(ii) exposing said engineered cyanobacterium to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said engineered cynanobacterium into n-alkanes, wherein the predominant n-alkane is n-pentadecane.

2. The method of claim 1, wherein the amount of said n-alkanes produced is at least two times the amount produced by an otherwise identical cyanobacterium, cultured under identical conditions, but lacking said recombinant acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes.

3. The method of claim 1, wherein said engineered cyanobacterium produces n-alkanes comprising both n-pentadecane and n-heptadecane, and wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 50%.

4. The method of claim 1, further comprising isolating n-pentadecane from said engineered cyanobacterium or said culture medium.

5. The method of claim 1, wherein said enzymes are encoded by a plasmid.

6. The method of claim 1 wherein said enzymes are encoded by recombinant genes incorporated into the genome of said engineered cyanobacterium.

7. The method of claim 1 wherein said enzymes are encoded by genes which are present in multiple copies in said engineered cyanobacterium.

8. The method of claim 1, wherein said acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes are at least 95% identical to SEQ ID NO: 10 and SEQ ID NO: 12, respectively.

9. The method of claim 1, wherein said acyl-ACP reductase enzyme and said recombinant alkanal decarboxylative monooxygenase enzyme are encoded by genes which are part of an operon, and wherein the expression of said genes is controlled by one or more inducible promoters.

10. The method of claim 9, wherein at least one promoter is a urea-repressible, nitrate-inducible promoter.

11. The method of claim 10, wherein said urea-repressible, nitrate-inducible promoter is P(nir07).

12. The method of claim 1, wherein said recombinant acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes are least 95% identical to SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

13. The method of claim 1, wherein said engineered cyanobacterium comprises at least two operons encoding distinct alkanal decarboxylative monooxygenase and acyl-ACP reductase enzymes.

14. The method of claim 13, wherein at least one operon encodes acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes which are at least 95% identical to SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

15. The method of claim 13, wherein at least one operon encodes acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes which are at least 95% identical to SEQ ID NO:10 and SEQ ID NO: 12, respectively.

16. The method of claim 6, wherein said acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes are at least 95% identical to SEQ ID NO:10 and SEQ ID NO:12, respectively.

17. The method of claim 16, wherein expression of said acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes is controlled by an inducible promoter.

18. The method of claim 17, wherein said engineered cyanobacterium produces at least 0.5% DCW n-alkanes in the presence of an inducer.

19. The method of claim 17, wherein said engineered cyanobacterium further comprises a second operon encoding acyl-ACP reductase and alkanal decarboxylative monooxygenase enzymes which are at least 95% identical to SEQ ID NO: 27 and SEQ ID NO: 29, respectively.

20. The method of claim 1, wherein at least 95% of said n-alkanes, by mass, are n-pentadecane and n-heptadecane, and wherein the percentage by mass of n-pentadecane relative to n-pentadecane plus n-heptadecane is at least 80%.