# Patent

## Title
Industrial fatty acid engineering general system for modifying fatty acids

## Abstract

Compositions and methods for a hybrid biological and chemical process utilizing chemotrophic microorganisms that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more desaturated hydrocarbons, unsaturated fatty acids, hydroxy acids, or diacids.

## Background
RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/536,056, filed Sep. 19, 2011 and entitled “INDUSTRIAL FATTY ACID ENGINEERING SYSTEM.” This application is also a continuation-in-part of International Patent Application No. PCT/US2011/34218, filed Apr. 27, 2011, and entitled “USE OF OXYHYDROGEN MICROORGANISMS FOR NON-PHOTOSYNTHETIC CARBON CAPTURE AND CONVERSION OF INORGANIC AND/OR C1-CARBON SOURCES INTO USEFUL ORGANIC COMPOUNDS,” which is a continuation-in-part of International Patent Application No. PCT/US2010/001402, filed May 12, 2010, and entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGANISMS FOR THE CHEMOSYNTHETIC FIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTO ORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS,” which is a continuation-in-part of U.S. patent application Ser. No. 12/613,550, filed Nov. 6, 2009, and entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGANISMS FOR THE CHEMOSYNTHETIC FIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTO ORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS,” which claims the benefit of U.S. Provisional Patent Application No. 61/111,794, filed Nov. 6, 2008, and entitled, “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGANISMS FOR THE RECYCLING OF CARBON FROM CARBON DIOXIDE AND OTHER INORGANIC CARBON SOURCES THROUGH CHEMOSYNTHESIS INTO BIOFUEL AND ADDITIONAL USEFUL PRODUCTS.” This application also claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/542,823, filed Oct. 4, 2011 and entitled “ENGINEERED CO2-FIXING CHEMOTROPHIC MICROORGANISMS PRODUCING CARBON-BASED PRODUCTS AND METHODS OF USING THE SAME.” This application also claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/616,560, filed Mar. 28, 2012 and entitled “PROCESS FOR GENERATING HYDROXYLATED FATTY ACIDS.” Each of these applications is incorporated herein by reference in its entirety for all purposes.
SEQUENCE LISTING
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 1, 2014, is named 164185.P001U1 SL.txt and is 146,662 bytes in size.


FIELD OF INVENTION
This disclosure relates to compositions capable of producing and methods of the producing oils, fuels, and oleochemicals through cultivating bacteria that grow on carbon-containing gas such as syngas, producer gas, CO2, carbon monoxide and mixtures of the same containing hydrogen gas. This disclosure further relates to methods of fixing carbon from gas input into useful organic molecules such as diacids, hydroxy fatty acids, unsaturated fatty acids. The bacteria of the invention can be genetically engineered for use in the methods or other aspects of the invention described herein.
BACKGROUND
Sustainable and renewable sources of organic molecules are needed to help reduce the amount of carbon dioxide emissions in the atmosphere, as well as to reduce global energy consumption based upon coal, oil, and natural gas economies. Increased demand in the global economy has placed increasing pressure on traditional hydrocarbon feedstocks for the production of organic molecules. Many industries, including plastics and chemical manufacturers, rely heavily on the availability of fossil hydrocarbon sources as a feedstock for their manufacturing processes. Cost-effective alternatives to current sources of supply could help mitigate the upward pressure on fossil resource demand and raw material costs.
Biologic systems that fix carbon through natural biochemical metabolic processes are known. Algal systems have been developed to create hydrocarbon or petroleum replacements through photosynthetic reactions, as well as heterotrophic reactions utilizing fixed carbon feedstocks such as sugar that indirectly depend upon photosynthesis, however insufficient yields limit the effectiveness, economic feasibility, practicality and commercial adoption of these algal technologies. Bacterial cells have also been genetically engineered to process sugar feedstocks into useful organic compounds in heterotrophic fermentation systems; however, there are significant drawbacks for these systems as well.
Heterotrophic fermentations are vulnerable to contamination because heterotrophic microorganisms that can grow on fixed carbon nutrients and compete with a production strain are far more ubiquitous in the surface environment. Heterotrophic technologies also generally suffer limitations in terms of food versus fuel conflict and negative environmental impacts.
Gas-to-liquid (GTL) technologies have the benefit of allowing the utilization of waste carbon sources—including highly lignocellulosic waste through the conversion to synthesis gas (syngas) via gasification, as well as waste CO2 through the provision of reduced hydrogen—in the production of organic chemicals. Syngas is a mix of gases that generally contains H.sub.2, CO, and CO.sub.2 as major components, which can be generated through steam reforming of methane and/or liquid petroleum gas or biogas or through gasification of any organic material, including but not limited to biomass, waste organic matter, various polymers, and coal. Many gasification processes are available for the production of syngas. A number of gasification processes subject the carbonaceous feedstock to partial oxidation at high temperatures (500-1500.degree. C.), with the oxygen supply restricted to prevent complete combustion, producing syngas with varying composition depending on feedstock and reaction conditions such that the ratio of H.sub.2:CO can range from 0.5:1 to 3:1. The hydrogen component of syngas can be raised through the reaction of CO with steam in the water gas shift reaction with a concomitant increase in CO.sub.2 in the syngas mix.
Some major technologies for syngas conversion to chemicals include chemical catalytic processes such as the Fischer-Tropsch (F-T) as well as processes for the synthesis of methanol or other mixed alcohols, and biological syngas fermentation processes. F-T has been worked on for almost one hundred years and relies on metal-based, inorganic catalysts for the conversion of syngas into longer chain hydrocarbons. Difficulties with F-T include: a wide chain length distribution of products resulting in the need to reprocess short chain length products such as methane and LPG and/or the need to perform additional costly post-processing steps on long chain waxes and tars such as hydrocracking; high catalyst sensitivity to syngas impurities such as sulfur containing compounds, tars, and particulates, generally necessitating multiple costly gas clean up steps; relatively low flexibility in terms of accommodating various ratios of syngas constituents i.e. H2:CO, and low tolerance of CO.sub.2, often resulting in additional costly syngas conditioning steps such as water gas shift and CO.sub.2 removal; the actual F-T step being relatively high temperature and pressure resulting in costly compression and heating requirements; the wide distribution of products generally necessitating the storage, handling, and transport of a wide array of products which is often uneconomic except for relatively large scale operations; F-T products (e.g. diesel, jet fuel, naphtha, waxes) being relatively low in value at current (2011) prices compared to many different higher value oils, lipids, and oleochemicals that can be produced biologically. The difficulties with F-T generally also apply to other chemical conversion processes such as methanol synthesis.
The gasification of biomass to generate syngas has a long history going back to World War II where biomass gasification was used for running modified automobiles, boats, buses, and trucks. Presently, a number of biomass gasification technologies are at, or near commercialization (able to gasify 10,000 or more tons of biomass per year), and are generally used for the production of heat and/or electricity. The synthesis of chemicals or fuels from syngas generated via biomass gasification is at an earlier stage of development, and is generally pre-commercial.
Using syngas and/or CO.sub.2 and/or renewable H.sub.2 in a gas bioprocess enables the utilization of cheaper and more flexible sources of energy and/or carbon for the biological synthesis of sustainable chemicals and fuels than is possible through heterotrophic or phototrophic synthesis. In a syngas bioprocess, syngas acts as both a carbon and energy source for the microbial culture. Some of the advantages of syngas bioprocesses include: the production of a relatively narrow range of carbon chain length distribution compared to F-T; lower sensitivity to syngas impurities; greater tolerance of varying ratios of H2:CO and the presence of CO2; ability to operate at much closer to ambient temperature and pressure; ability to produce various higher value oleochemical products.
A bioprocess based upon a gaseous feedstock such as syngas can allow for far lower negative environmental and food production impacts in the biological synthesis of liquid fuels and/or chemicals than the highly land and water intensive heterotrophic or phototrophic-based technologies. However, current biological GTL technologies generally yield relatively short chain alcohols, or other short chain organic compounds, as products, which have relatively low energy density and infrastructure compatibility and limited applications. The syngas-growing microorganisms used in current biological GTL technologies are generally inappropriate for the synthesis of mid- to long-carbon chain lipid-based chemicals. Furthermore the types of microorganisms used in current biological GTL technologies such as Clostridia have a relatively low tolerance for their short carbon chain gas fermentation products such as ethanol, butanol, or acetic acid, which limits titers and complicates product recovery, hurting the overall economics of the GTL process.
There is a need to identify a set of microorganisms that can grow in conventional and scalable contained reaction vessels and that produce commercially viable sets of organic carbon chains of at least eight carbon atoms long in a commercially feasible method. There is a need to identify microorganisms not limited metabolically by typical fixed carbon inputs such as sugar, and a microorganism that can additionally utilize syngas, producer gas, as well as a wide array of abiotic sources of carbon and energy for the synthesis of drop-in fuels and chemicals, leading to a feedstock flexibility that far exceeds comparable heterotrophic systems. There is a need to identify and use microorganisms that can utilize electron donors such as hydrogen, present in syngas, producer gas, as well as readily generated through a wide array of abiotic renewable energy technologies, for growth and carbon fixation.
The targeting of fatty acids produced through fatty acid biosynthesis to short chain lengths has been achieved in heterotrophic microorganisms. This has been accomplished through the use of thioesterases to change populations of fatty acids C8-C14 and the over-expression of thioesterases to increase shorter chain length fatty acids.
Examples in the prior art include C8-C14 thioesterase expression to produce shorter chain lengths in U.S. Pat. No. 7,883,882 Renewable chemical production from novel fatty acid feedstocks, Franklin et al. Solazyme, p. 58.
However there is a need to target the production of shorter chain length fatty acids in microorganisms that are capable of growing and producing lipids chemotrophically on syngas or H.sub.2/CO.sub.2 gas mixes to enable microbial GTL production of lipids with targeted, mid-length carbon chains.
Dicarboxylic acids (Diacids) such as dodecanedoic acid (n=10) are used in production of nylon (nylon-6,12), polyamides, coatings, adhesives, greases, polyesters, dyes, detergents, flame retardants and fragrances. Diacids can be produced by fermentation of long-chain alkanes by candida tropicalis (Kroha K, Infom 2004, 15, 568). Traumatic acid, monounsaturated dodecanedoic acid (10E-dodeca-1,12-dicarboxylic acid) has been produced from plant tissues English J et al., Science 1939, 90, 329. Pyrococcus furiosus produces an array of dicarboxylic acids (Carballeira, 1997). The total amount of dicarboxylic acids comprises only 3.4% of the total, however, this could be boosted by various literature methods.
There is a need for a biological, non-heterotrophic means of producing diacids from low-cost or sustainable syngas feedstocks.
Nutritionally important n-3 fatty acids include α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), all of which are polyunsaturated. N-3 fatty acids that are important in human physiology are α-linolenic acid (18:3, n-3; ALA), eicosapentaenoic acid (20:5, n-3; EPA), and docosahexaenoic acid (22:6, n-3; DHA). These three polyunsaturates have either 3, 5, or 6 double bonds in a carbon chain of 18, 20, or 22 carbon atoms, respectively. As with most naturally produced fatty acids, all double bonds are in the cis-configuration.
A fatty acid desaturase is an enzyme that removes two hydrogen atoms from a fatty acid, creating a carbon/carbon double bond. These desaturases are classified as delta—indicating that the double bond is created at a fixed position from the carboxyl group of a fatty acid (for example, Δ9 desaturase creates a double bond at the 9th position from the carboxyl end). omega (e.g. ω3desaturase)—indicating the double bond is created between the third and fourth carbon from the methyl end of the fatty acid. In the biosynthesis of essential fatty acids, an elongase alternates with different desaturases (for example, Δ6desaturase) repeatedly inserting an ethyl group, then forming a double bond.
Most polyunsaturated oils come from fish and there is a need for alternate, and particularly microbial sources of polyunsaturated fatty acids, given depleting fish stocks and increasing pollution in the oceans.
SUMMARY OF THE INVENTION
The present invention allows microorganisms to be engineered to convert CO.sub.2 gas and/or syngas and/or producer gas to higher value mid- to long-carbon chain length oleochemicals or monomers. The present technology allows the development of new genetically enhanced strains of microorganisms that can be used for syngas bioprocessing within biological gas-to-liquid (GTL) processes to produce and/or secrete various relatively long chain organic compounds that are drop-in, and are currently only produced in bulk from petroleum or higher plants.
The present invention relates to the engineering of microorganisms, including but not limited to hydrogen oxidizing, carbon monoxide oxidizing, and knallgas microorganisms, with a natural capability to grow and synthesize biomass on gaseous carbon sources such as syngas and/or CO.sub.2, such that the engineered microorganisms synthesize targeted products, including chemicals and fuels, under gas cultivation. The microorganisms and methods of the present invention enable low cost synthesis of chemicals and fuels, which can compete on price with petrochemicals and higher-plant derived oleochemicals and monomers, and which will generally have a substantially lower price than oleochemicals produced through heterotrophic or phototrophic synthesis.
The invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more lipids. In some embodiments, the composition comprises a microorganism, wherein the microorganism is a hydrogen-oxidizing chemotrophic microorganism. In some embodiments, the composition comprises a microorganism, wherein the microorganism is a carbon monoxide-oxidizing microorganism. In some embodiments, the composition comprises a microorganism, wherein the microorganism is a knallgas microorganism. In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Rhodococcus or Gordonia. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205) or Rhodococcus sp (DSM 3346). In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator. 
In some embodiments, the composition comprises a microorganism wherein the microorganism can naturally grow on H2/CO2 and/or syngas, and wherein the microorganism can naturally accumulate lipid to 50% or more of the cell biomass by weight. In some embodiments the microorganisms have a native ability to send a high flux of carbon down the fatty acid biosynthesis pathway. In some embodiments the microorganism exhibiting these traits is Rhodococcus opacus (DSM 43205 or DSM 43206).
In some embodiments, the composition comprises a microorganism that can naturally grow on H2/CO2 and/or syngas, and wherein the microorganism can naturally accumulate polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) to 50% or more of the cell biomass by weight. In some embodiments the microorganisms have a native ability to direct a high flux of carbon through the acetyl-CoA metabolic intermediate, which can lead into fatty acid biosynthesis, along with a number of other synthetic pathways including PHA and PHB synthesis. A microorganism is considered to direct a high flux of carbon through acetyl-CoA if a product of a synthesis pathway going through the acetyl-CoA metabolic intermediate, including but not limited to polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA), can represent 50% or more of the cell biomass by weight. In some embodiments the microorganism exhibiting these traits is Cupriavidus necator (DSM 531 or DSM 541).
In some embodiments, the invention relates to a non-naturally occurring microorganism capable of converting syngas or other gaseous carbon sources into targeted oleochemical and/or monomer products, where the wild-type microorganism is capable of growing on syngas or other gaseous carbon sources, but is either not capable of synthesizing said targeted oleochemical and/or monomer products, or is capable of synthesizing the targeted oleochemicals and/or monomers, but is not capable of synthesizing the targeted biochemical products at the concentration and/or efficiency of the non-natural microorganism. In such microorganisms, one or more proteins or enzymes are expressed in the microorganism, thereby modifying, extending, diverting, enhancing, promoting, or otherwise altering the lipid biosynthesis pathway or its regulation for the synthesis and/or enhanced synthesis of a targeted lipid-based product, oleochemical, monomer, or hydrocarbon.
In some embodiments, the invention relates to a non-naturally occurring microorganism capable of converting syngas or other gaseous carbon sources into targeted oleochemical and monomer products, where the wild-type microorganism is capable of growing on syngas or other gaseous carbon sources and is capable of synthesizing said targeted oleochemical and monomer products, but the non-naturally occurring microorganism is capable of synthesizing the targeted biochemical products at a higher concentration and/or efficiency than the wild-type microorganism due to the overexpression and/or underexpression of one or more proteins or enzymes.
In some embodiments, the invention relates to compositions comprising one or more bacterial cells that consist of one, two, or three exogenous nucleic acid sequences where said bacteria can grow using syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas as a source of carbon and/or energy.
In some embodiments, the invention relates to compositions comprising one or more bacterial cells of Rhodococcus opacus (DSM 43205) that consist of zero, one, two, or three exogenous nucleic acid sequences.
In some embodiments one, two, or three exogenous nucleic acid sequences encode one or more thioesterase proteins.
In some embodiments the source of thioesterase is inherent to the production organisms. In some embodiments the source of thioesterase is Rhodococcus opacus B4. In some embodiments the thioesterase is derived from bacteria or plants other than the host microorganism.
In some embodiments, the invention relates to compositions comprising one or more bacterial cells that consist of two exogenous nucleic acid sequences that encode the following proteins: fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase, where said bacteria can grow using syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas as a source of carbon and/or energy.
In some embodiments, the invention relates to compositions comprising one or more bacterial cells that consist of three exogenous nucleic acid sequences that encode the following proteins: fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase, and a thioesterase, where said bacteria can grow using syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas as a source of carbon and/or energy.
In some embodiments, the bacterial cell produces and/or secretes one or more lipids in an amount that is greater than the amount of lipids produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
In some embodiments, the bacterial cell produces and/or secretes one or more lipids having a given carbon chain length, where the amount of said lipid produced and/or secreted is greater than the amount produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
In some embodiments, the bacterial cell produces and/or secretes one or more lipid molecules in an amount that is less than the amount of lipids produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
In some embodiments, the bacterial cell produces and/or secretes one or more hydrocarbons in an amount that is greater than the amount of hydrocarbons produced and/or secreted by the same cell not comprising the exogenous nucleic acid sequence.
In some embodiments, the bacterial cell or compositions comprising the bacterial cell comprise at least one exogenous nucleic acid sequence that is integrated into the genome of the cell.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase. In some embodiments the microorganism is Rhodococcus opacus. 
In some embodiments the invention relates to a method of producing a lipid or mixture of lipids in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In some embodiments, the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas. In some embodiments, the microorganism population comprises a bacterial strain of Rhodococcus opacus. In some embodiments, the microorganism population comprises a bacterial strain of Rhodococcus opacus (DSM 43205 or 43206).
In some embodiments, the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising methanol, a common impurity of syngas, with or without the addition of syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas. In some embodiments, the microorganism population comprises a bacterial strain of Rhodococcus opacus. In some embodiments, the microorganism population comprises a bacterial strain of Rhodococcus opacus (DSM 43205).
In some embodiments, the invention relates to a method of producing a lipid or mixture of lipids, wherein the method comprises: culturing a population of bacterial cells comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas. In some embodiments, the microorganism population comprises a bacterial strain of Cupriavidus necator. 
In some embodiments, the molecule is chosen from one or more alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or dicarboxylic acid (diacid). In some embodiments, the method produces a lipid or mixture of lipids at a quantity higher than the quantity of lipid or mixture of lipids in the same bacterial cell population not comprising the exogenous nucleic acids described herein. In some embodiments the one or more lipids comprise a quantity of at least one alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid at a level higher than the quantity of the alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid in the same microorganism not comprising the exogenous nucleic acid sequences.
In some embodiments of the invention, the invention relates to a method of producing and/or secreting a lipid or mixture of lipids by culturing a population of microorganisms comprising a bacterial cell described herein, wherein the exogenous nucleic acid sequences are operably linked to a promoter that is inducible in response to a first stimulus, and wherein the method further comprises: culturing the population of bacterial cells for a first period of time in the presence of a first stimulus to produce one or more lipids chosen from an alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid.
In some embodiments, the invention relates to a method of producing one or more diacids, comprising exposing a bacterial cell to syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2; wherein the bacterial cell is capable of fixing gaseous CO2 into one or more diacids, and wherein the microorganism comprises at least a first exogenous nucleic acid and a second exogenous nucleic acid, wherein the first exogenous nucleic acid encodes cytochrome P450 enzyme and the second exogenous nucleic acid encodes NADPH cytochrome P450 reductase or NADH cytochrome P450 reductase. In some embodiments, the first and second exogenous nucleic acids are heterologous nucleic acid sequences. In some embodiments, the bacterial cell comprises at least a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a cytochrome P450 enzyme, the second exogenous nucleic acid sequence encodes a NADPH cytochrome P450 reductase, and the third exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the bacterial cell comprises at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acid sequences that encode a lipid pathway enzyme. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346). In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator. In some embodiments the microorganism is from the suborder corynebacterineae or the family burkholderiaceae. In some embodiments the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
In some embodiments, the invention relates to a method of producing one or more hydroxyacid, diacid, or unsaturated fatty acid, or any combination thereof comprising exposing a bacterial cell to syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2; wherein the bacterial cell is capable of fixing gaseous CO2 into one or more lipids; wherein the lipids are recovered from the bioreactor and fed to a second bioreactor wherein the lipids are postprocessed to generate hydroxyacid, diacid, and/or unsaturated fatty acids via a second microorganism such as but not limited to Candida tropicalis. In some embodiments, the bacterial cell comprises at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the composition comprises a bacterial cell, wherein the bacteria is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346). In some embodiments, the bacterial cell is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the bacterial cell is Cupriavidus necator. In some embodiments the bacterial cell is from the suborder corynebacterineae or the family burkholderiaceae. In some embodiments the bacterial cell through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
In some embodiments, the invention relates to a method of producing one or more unsaturated fatty acids, comprising exposing a bacterial cell to syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2; wherein the bacterial cell is capable of fixing gaseous CO2 into one or more unsaturated fatty acids and wherein the microorganism comprises at least a first exogenous nucleic acid, wherein the first exogenous nucleic acid encodes a desaturase that introduces double bonds to fatty acids. In some embodiments, the first exogenous nucleic acids is a heterologous nucleic acid sequence. In some embodiments, the bacterial cell comprises at least a first, and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a desaturase, the second exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346). In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator. In some embodiments the microorganism is from the suborder corynebacterineae or the family burkholderiaceae. In some embodiments the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
In some embodiments, the invention relates to a method of producing one or more hydroxy fatty acids (hydroxy acids), comprising exposing a bacterial cell to syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2; wherein the bacterial cell is capable of fixing gaseous CO2 into one or more hydroxy acids and wherein the microorganism comprises at least a first exogenous nucleic acid, wherein the first exogenous nucleic acid encodes a P450-dependent fatty acid hydroxylase that introduces hydroxyl groups at positions along the fatty acid chain. In some embodiments, the first exogenous nucleic acids is a heterologous nucleic acid sequence. In some embodiments, the bacterial cell comprises at least a first, and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a P450-dependent fatty acid hydroxylase, the second exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Rhodococcus opacus (DSM 43205 or 43206) or Rhodococcus sp (DSM 3346). In some embodiments, the composition comprises a microorganism, wherein the microorganism is chosen from the genera Ralstonia or Cupriavidus. In some embodiments, the composition comprises a microorganism, wherein the microorganism is Cupriavidus necator. In some embodiments the microorganism is from the suborder corynebacterineae or the family burkholderiaceae. In some embodiments the microorganism through its native machinery produces a complement of fatty acids described in the Fatty Acid Output section below.
In some embodiments, the invention relates to a method of manufacturing one or more lipids, comprising (a) culturing a cell described herein in a reaction vessel or bioreactor in the presence of syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2, wherein the cell produces and/or secretes one or more lipids in an quantity equal to or greater than at least 10% of the cell's total dry cellular mass; and (b) separating the one or more lipids from reaction vessel. In some embodiments, the method further comprises purifying the one or more lipids after separation from the reaction vessel or bioreactor.
In some embodiments, the invention relates to a method of producing a alkene or alkyne in a bacterial cell comprising at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes a fatty acid aldehyde decarbonylase.
In some embodiments, the bacterial cell producing a alkene or alkyne comprises at least a first, a second, and a third exogenous nucleic acid sequences, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid encodes a thioesterase.
In some embodiments, the invention relates to a bioreactor comprising the composition or bacterial cells described herein.
In some embodiments, the invention relates to a system for the production of one or more lipids or mixture of lipids, comprising a bioreactor, which comprises: (a) a microorganism population comprising a cell described herein; and (b) an inlet connected to a feedstock source allowing delivery of a feedstock comprising syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2.
In some embodiments, the invention relates to the population of fatty acids being modified to produce molecules of desired carbon chain length by incorporation of one or more thioesterases.
In some embodiments, the invention relates to the population of fatty acids being modified to add additional carboxylic acid (—COOH) groups using exogenous enzymes.
In some embodiments, the invention relates to the population of fatty acids being modified to add hydroxyl groups (—OH) using the exogenous enzymes (hydroxylases).
In some embodiments, the invention relates to the population of fatty acids being modified to add desaturation through the incorporation of one or more double bonds, using the exogenous enzymes (desaturases).



BRIEF DESCRIPTION OF THE DRAWINGS
Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:
FIG. 1 describes the taxonomic names afforded to the chemoautotrophic and oleaginous microorganisms used in selected embodiments of the invention.
FIG. 2 shows the 16S rRNA gene based-rooted phylogenetic tree of gordoniaceae, mycobacteriaceae, nocardiaceae and burkholderiaceae.
Bar, 0.01% estimated sequence divergence.
FIG. 3 shows the sequence similarity of Rhodococcus opacus (DSM 43205) 16S rRNA gene (NR_026186.1) to members of the family gordoniaceae, mycobacteriaceae, nocardiaceae and burkholderiaceae. The Genbank accession numbers, DNA length and % identity of analyzed genes are indicated.
FIG. 4 describes the nucleotide sequence alignment of the 16S rRNA genes. Alignment discloses SEQ ID Nos 40, 12-34, 41, and 3539, respectively, in order of appearance.
FIG. 5 demonstrates the growth of chemotrophic and oleaginous microorganisms on different carbon sources. Bacterial growth was measured using optical density (OD) detection at 650 nm after the indicated days (in parentheses). Media and growth conditions described in the Examples section below. ND, not done.
FIG. 6 describes the measured lipid content of microorganisms on heterotrophic and chemoautotrophic growth conditions as a percentage of total cellular dry matter (CDM). Cells were grown under conditions described in FIG. 5, harvested after 72 hr (unless otherwise indicated) and analyzed by gas chromatography. For CDM, total dry weight was determined gravimetrically.
FIG. 7 describes the fatty acid profile of R. opacus (DSM 44193) under heterotrophic growth conditions. Cells were harvested after 72 hr and analyzed by gas chromatography.
FIG. 8 describes the fatty acid profile R. opacus (DSM43205) under heterotrophic (A) and chemoautotrophic (B) growth conditions. Cells were harvested after 72 hours of growth and analyzed by gas chromatography.
FIG. 9 describes the fatty acid profile Rhodococcus sp. (DSM 3346) under heterotrophic (A) chemoautotrophic (B) growth conditions. Cells were harvested after 72 hr and analyzed by gas chromatography.
FIG. 10 describes shuttle vectors (A) and genetic elements (B) for transformation and gene expression of in chemoautotrophic and oleaginous microorganisms. MCS: multiple cloning site.
FIG. 11 describes the map of the plasmids pSeqCO1 (A; SEQ ID: 01), pSeqCO2 (B; SEQ ID: 02), pVer1 (C; SEQ ID: 03) and pVer2 (D; SEQ ID: 04) described in FIG. 10. The genetic elements are indicated.
FIG. 12 describes the transformation of chemoautotrophic and oleaginous microorganisms with shuttle vectors described in FIG. 10.
FIG. 13 describes the growth of Cupriavidus necator (DSM531) transformed with the plasmid (Y) pSeqCO2 (SEQ ID:2) and untransformed (N) on different kanamycin concentrations. Single colony of transformants and control were grown LB medium (per 1 L: 10 g Bacto-tryptone, 5 g yeast extract, 10 g NaCl pH=7.0) at 30° C. in the indicated kanamycin concentrations. The growth was measured using O.D650 after the indicated number of days.
FIG. 14 describes the restriction map related to the cloning strategy of FadDR and Fad genes into pSeqCO2 plasmid transformed for the experiment. Genes from the cyanobacterium (Synechocystis sp. PCC 6803) used in the experiment were FadR (SEQ ID: 08) and FAD (SEQ ID: 09) driven by the Synechocystis sp. Rubisco large subunit promoter (SEQ ID: 10) were cloned into pSeqCO2 plasmid using the indicated restriction sites to give pSeqCO2::FUEL.
FIG. 15 describes the production of hydrocarbons in Cupriavidus necator transformed with pSeqCO2::FUEL (Cn_FUEL2.1) and empty vector (Cn-P). GC chromatogram of hydrocarbon (indicated in red) extracted from transformants grown in 50 ml LB media under previously identified conditions.
FIG. 16 describes the hydrocarbons specific products and distribution (percentage in parentheses) from Cupriavidus necator transformed with pSeqCO2::FUEL (Cn_FUEL2.1 and Cn_FUEL2.2) and empty vector (Cn-P).
FIG. 17 shows the molecular structure of two hydrocarbons that were produced in relative abundance in the Cupriavidus necator transformed with pSeqCO2::FUEL (Cn_FUEL2.1 and Cn_FUEL2.2).
FIG. 18 describes the effect of pSeqCO2::FUEL (Cn_FUEL2.1 and 2.2) and empty vector (Cn-P) on the fatty acids distribution under the experimental conditions described previously.
FIG. 19 describes the modification of the fatty acid chain length by the enzymatic action of thioesterase (TE) in oleaginous bacteria.
FIG. 20 describes the modification of the fatty acid chain length by the enzymatic action of fatty acyl-ACP thioesterase (TE) in burkholderiaceae.
FIG. 21 describes the similarity of Rhodococcus opacus (B4) thioesterases protein sequence (YP_002784058.1) to other organisms. The Genbank accession numbers, amino acid length and % identity of analyzed proteins are indicated.
FIG. 22 describes the chemoautotrophic growth of Cupriavidus necator transformed with pSeqCO2::FUEL (Cn-FUEL2.1), empty vector (Cn-P) and untransformed (Cn). Bacterial growth was measured at O.D650 after 12 days.



DETAILED DESCRIPTION
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
The terms “amino acid” refer to a molecule containing both an amine group and a carboxyl group that are bound to a carbon, which is, designated the α-carbon. Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. In some embodiments, a single “amino acid” might have multiple sidechain moieties, as available per an extended aliphatic or aromatic backbone scaffold. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.
The term “biomass” refers to a material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material, includes, but is not limited to, compounds secreted by a cell.
The term “bioreactor” or “fermentor” refers to a closed or partially closed vessel in which cells are grown and maintained. The cells may be, but are not necessarily held in liquid suspension. In some embodiments rather than being held in liquid suspension, cells may alternatively be growing and/or maintained in contact with, on, or within another non-liquid substrate including but not limited to a solid growth support material.
The term “catalyst” refers to a chemical actor, such as a molecule or macromolecular structure, which accelerates the speed at which a chemical reaction occurs where a reactant or reactants is converted into a product or products, while the catalyst is not turned into a product itself, or otherwise changed or consumed at the completion of the chemical reaction. After a catalyst participates in one chemical reaction, because it is unchanged, it may participate in further chemical reactions, acting on additional reactants to create additional products. To accelerate a chemical reaction a catalyst decreases the activation energy barrier across the reaction path allowing it to occur at a colder temperature, or faster at a given temperature. In this way a more rapid approach of the system to chemical equilibrium may be achieved. Catalysts subsume enzymes, which are protein catalysts.
The term “cellulosic material” refers to any material with a high amount of cellulose, which is a polysaccharide having the formula (C6H10O5)n, that generally consists of a linear chain of hundreds to thousands of β(1→4) linked D-glucose monomers. Sources of cellulosic material include but are not limited to cardboard, cotton, corn stover, paper, lumber chips, sawdust, sugar beet pulp, sugar cane bagasses, and switchgrass.
The term “CoA” or “coenzyme A” refers to an organic cofactor for condensing enzymes involved in fatty acid synthesis and oxidation, pyruvate oxidation, acetyl or other acyl group transfer, and in other acetylation.
The term “cofactor” subsumes all molecules needed by an enzyme to perform its catalytic activity. In some embodiments, the cofactor is any molecule apart from the substrate.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C, H), nonpolar side chains (e.g., G, A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in an amino acid sequence encoded by an exogenous nucleic acid sequence, for example, is replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other biochemical properties (e.g. 2-thienylalanine for phenylalanine).
As used herein, “enzyme fragment” is meant to refer to a fragment of an enzyme that includes the sequences sufficient to function substantially similar to the function of the wild-type enzyme upon which the fragment sequence is based. Fragments are generally 10 or more amino acids in length. Some preferred lengths of fatty acid reductase are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220, at least 225, least 230 at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, or at least 430. Some preferred lengths of fatty acid reductase fragments are 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer, 230 or fewer, 235 or fewer, 240 or fewer, 245 or fewer, 250 or fewer, 255 or fewer, 260 or fewer, 265 or fewer, 270 or fewer, 275 or fewer, 280 or fewer, 285 or fewer, 290 or fewer, 295 or fewer, 300 or fewer, 305 or fewer, 310 or fewer, 315 or fewer, 320 or fewer, 325 or fewer, 330 or fewer, 335 or fewer, 340 or fewer, 345 or fewer, 350 or fewer, 355 or fewer, 360 or fewer, 365 or fewer, 370 or fewer, 375 or fewer, 380 or fewer, 385 or fewer, 390 or fewer, 395 or fewer, 400 or fewer, 415 or fewer, 420 or fewer, 425 or fewer, 430 or fewer, or 435 or fewer. Some preferred lengths of fatty acid decarbonylase are at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220, at least 225, least 230 at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, or at least 420 amino acids long. In some embodiments, the lengths of the fatty acid decarbonylase fragments are 15 or fewer, amino acids, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer, 230 or fewer, 235 or fewer, 240 or fewer, 245 or fewer, 250 or fewer, 255 or fewer, 260 or fewer, 265 or fewer, 270 or fewer, 275 or fewer, 280 or fewer, 285 or fewer, 290 or fewer, 295 or fewer, 300 or fewer, 305 or fewer, 310 or fewer, 315 or fewer, 320 or fewer, 325 or fewer, 330 or fewer, 335 or fewer, 340 or fewer, 345 or fewer, 350 or fewer, 355 or fewer, 360 or fewer, 365 or fewer, 370 or fewer, 375 or fewer, 380 or fewer, 385 or fewer, 390 or fewer, 395 or fewer, 400 or fewer, 415 or fewer, 422 or fewer. Some preferred lengths of thioesterase fragments are at least 10 amino acids, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210 at least 215, at least 220, at least 225, least 230 at least 235, at least 240, at least 245, at least 250 or at least 255. Some preferred lengths of thioesterase fragments are 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer, 40 or fewer, 45 or fewer, 50 or fewer, 55 or fewer, 60 or fewer, 65 or fewer, 70 or fewer, 75 or fewer, 80 or fewer, 85 or fewer, 90 or fewer, 95 or fewer, 100 or fewer, 105 or fewer, 110 or fewer, 115 or fewer, 120 or fewer, 125 or fewer, 130 or fewer, 135 or fewer, 140 or fewer, 145 or fewer, 150 or fewer, 155 or fewer, 160 or fewer, 165 or fewer, 170 or fewer, 175 or fewer, 180 or fewer, 185 or fewer, 190 or fewer, 195 or fewer, 200 or fewer, 205 or fewer, 210 or fewer, 215 or fewer, 220 or fewer, 225 or fewer, 230 or fewer, 235 or fewer, 240 or fewer, 245 or fewer, 250 or fewer, 255 or fewer or 260 or fewer. As used in the paragraph herein reference to preferred fragment sizes are intended to refer to all permutation of ranges between at least and less than such as ranges may be any number set forth as an “at least” size to any number set forth as an “less than t” size in order to provide a range of sizes such as 20-400, 20-30, 40-100, etc.
The terms “exogenous gene” means a nucleic acid that has been recombinantly introduced into a cell, which encodes the synthesis of RNA and/or protein. In some embodiments, the exogenous gene is introduced by transformation. In some embodiments, the exogenous gene is introduced into the cell by electroporation. A transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene put into the host species may be taken from a different species (this is called heterologous), or it may naturally occur within the same species (this is homologous as defined below). Therefore, exogenous genes subsume homologous genes that are integrated within or introduced to regions of the genome, episome, or plasmid that differ from the locations where the gene naturally occurs. Multiple copies of the exogenous gene may be introduced into the cell. An exogenous gene may be present in more than one copy within the host cell or transformed cell. In some embodiments, the microorganism comprises between and including 1 and 10,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 1,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 10,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 1,000 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the microorganism comprises between and including 1 and 500 copies of the nucleic acid that encodes an exogenous protein. In some embodiments, the exogenous gene is maintained by a cell as an insertion into the genome or as an episomal molecule. In some embodiments, the microorganism comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1000 copies of the one or more nucleic acids that encode one or more exogenous proteins.
As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operably linked to a coding sequence that encodes an enzyme or fragment thereof capable of conferring enzymatic activity to a cell, such that when present in the cell, the coding sequence will be expressed. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than ten expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than nine expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than eight expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than seven expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than six expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than five expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than four expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than three expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than two expressible forms of exogenous nucleic acid sequences. In some embodiments of the invention, the composition comprising the microorganisms or bacterial cells of the present invention comprise no more than one expressible form of an exogenous nucleic acid sequences.
SEQ ID NO:1 refers to Sequesco plasmid sequence 1.
SEQ ID NO:2 refers to Sequesco plasmid sequence 2.
SEQ ID NO: 3 refers to Sequesco plasmid Ver1 plasmid sequence.
SEQ ID NO:4 refers to Sequesco plasmid Ver2 plasmid sequence.
SEQ ID NO:5 refers to cyanobacterium FadR.
SEQ ID NO:6 refers to cyanobacterium FAD.
SEQ ID NO:7 refers to cyanobacterium Rubisco large subunit promoter
SEQ ID NO:8, refers to the 16S rRNA sequence from the genus Rhodococcus opacus DSM43205
SEQ ID NO:9 refers to the 16S rRNA sequence from the genus Rhodococcus opacus B4.
SEQ ID NO:10 refers to the 16S rRNA sequence from the genus Ralstonia. 
SEQ ID NO:11 refers to Rhodococcus opacus TE
The terms “fatty acyl-ACP thioesterase” (TE) mean an enzyme that catalyzes the cleavage of a fatty acid from an acyl carrier protein (ACP) during lipid synthesis.
The terms “fatty acyl-ACP/acyl-CoA reductase” (FadR) refers to an enzyme catalyzing the reaction that produces a fatty aldehyde from an acyl-ACP or acyl-CoA molecule by reduction.
The terms “fatty aldehyde decarbonylase” (FAD) refers to an enzyme catalyzing the reaction that produces an alkane from a fatty aldehyde molecule by decarbonylization.
As used herein, the term “functional fragment” is meant to refer to a fragment of any polypeptide or amino acid sequence that is encoded by an exogenous nucleic acid sequence of the present invention which retains its ability to function like the amino acid sequence to which the fragment is homologous. Functional fragments of enzymes are at least about 5 amino acids in length derived from enzyme and may comprise non-wild-type amino acid sequences. One having ordinary skill in the art can readily determine whether a protein or peptide is a functional fragment of a particular amino acid sequence by examining its sequence and testing its ability to function in a fashion similar to that function of the amino acid sequence upon which the fragment is based. Truncated versions of exogenous proteins may be prepared and tested using routine methods and readily available starting material. As used herein, the term “functional fragment” is also meant to refer to peptides, polypeptides, amino acid sequence linked by non-peptidal bonds, or proteins which comprise an amino acid sequence that is identical or substantially homologous to at least a portion of the exogenous amino acid sequence and which are capable of functioning in a similar function to the exogenous amino acid sequence to which the fragment is homologous. The term “substantially homologous” refers to an amino acid sequence that has conservative substitutions. One having ordinary skill in the art can produce functional fragments of the FadD, FAD, thioesterase, cytochrome P450 enzyme, desaturase, and hydroxylase amino acid sequences following the disclosure provided herein and well known techniques. The functional fragments thus identified may be used and formulated in place of full length FadD, FAD, thioesterase, cytochrome P450 enzyme, desaturase, and hydroxylase without undue experimentation.
The term “gasification” refers to a generally high temperature (>700° C.) process that converts carbonaceous materials into a mixture of gases including hydrogen, carbon monoxide, and carbon dioxide called syngas or producer gas. The process generally involves partial combustion and/or the application of externally generated heat along with the controlled addition of oxygen and/or steam.
As used herein, “homologous” refers to the sequences homology between two nucleic acid sequences or two amino acid sequences. Two nucleic acid sequences or two amino acid sequences that are sufficiently homologous to retain immunogenic function are “homologues.” Sequence homology for nucleotides and amino acids may be determined using FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP* 4.0b10 software (D. L. Swofford, Sinauer Associates, Massachusetts). “Percentage of similarity” is calculated using PAUP* 4.0b10 software (D. L. Swofford, Sinauer Associates, Massachusetts). The average similarity of the enzymatic sequence or 16S rRNA sequence is calculated compared to all sequences in the phylogenic tree. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403410, which is incorporated herein by reference in its entirety). Software for performing BLAST analyses is publicly available though the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
The term “hydrocarbon” refers to a molecule composed exclusively of carbon and hydrogen atoms with the carbons bonded covalently in a branched, cyclic, linear, or partially cyclic chain and with hydrogen atoms covalently bonded to the carbons such that the chemical octet rule for the carbons is generally satisfied. In some hydrocarbons there may occur some number of double or triple bonds between adjacent carbon atoms in the chain. Thus, the label hydrocarbon subsumes branched, cyclic, linear, branched, or partially cyclic alkanes (also called paraffins), alkenes (also called olefins), and alkynes. The structure of hydrocarbon molecules range from the smallest, methane (CH4), a primary component of natural gas, to high molecular weight complex molecules including asphaltenes present in bitumens crude oil, and petroleum. Other examples include dodecane (C12), hexadecane (C16), or octadecane (C18) etc. Hydrocarbons of the present invention may be in gaseous, liquid, or solid phases, either as singly or in multiply coexisting phases. In some embodiments, the hydrocarbons are selected from one or more of the following: linear, branched, cyclic, or partially cyclic alkanes, alkenes, alkynes, lipids, and paraffin. In some embodiments the hydrocarbon are selected from one or more of the following: octane, squalene Spiro[4.5]decane, Bicyclo[10.8.0]eicosane, cis,cis-1,6-Dimethylspiro[4.5]decane, 1,19-Eicosadiene, Cyclooctacosane, Bicyclo[10.8.0]eicosane, 1-Pentadecyne, 1-Pentadecyne, Heptacosyl acetate, 5-Cyclohexyl-1-pentene, 1-Hexadecyne and Cyclodecacyclotetradecene, -eicosahydro.
The term “hydrophobic fraction” gives the fraction of matter that has low solubility in water and greater solubility in a hydrophobic phase than in an aqueous phase. In some embodiments, the hydrophobic fraction is non-polar. In some embodiments, the genetically modified bacterial cells described herein increase the hydrophobic fraction of hydrocarbons in a cell as compared to the same cell that is not genetically modified.
The term “improve lipid yield” refers to an increase in the lipid production of an organism through any means. In some embodiments, the increase is caused by raising the cell dry weight density of a microbial culture and/or raising the fraction of cell mass that is composed of lipid and/or reducing the cell doubling time and/or the biomass doubling time, resulting in an overall increase in the lipid production rate per unit volume.
The term “knallgas” refers to the mixture of molecular hydrogen and oxygen gas. A “knallgas microorganism” is a microbe that can use hydrogen as an electron donor and oxygen as an electron acceptor in the generation of intracellular energy carriers such as Adenosine-5′-triphosphate (ATP). The terms “oxyhydrogen” and “oxyhydrogen microorganism” can be used synonymously with “knallgas” and “knallgas microorganism” respectively.
The term “lignocellulosic material” is any material composed of cellulose, hemicellulose, and lignin where the carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to lignin. Lignocellulosic materials subsume agricultural residues (including corn stover and sugarcane bagasse), most biomass energy crops, wood residues (including sawmill and paper mill discards), and a substantial fraction of municipal waste.
The terms “lipids” refers to category of molecules that can be dissolved in nonpolar solvents (such as chloroform and/or ether) and which also have low or no solubility in water. The hydrophobic character of lipids molecules typically results from the presence of long chain hydrocarbon sections within the molecule. Lipids subsume the following molecule types: hydrocarbons, fatty acids (saturated and unsaturated), fatty alcohols, fatty aldehydes, hydroxy acids, diacids, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, sterols such as cholesterol and steroid hormones, fat-soluble vitamins (such as vitamins A, D, E and K), polyketides, terpenoids, and waxes.
The term “lipid modification enzyme” corresponds to an enzyme that catalyzes a reaction changing a lipid's covalent bonds such as TE, FAR, FadR, FAD, fatty aldehyde reductase, lipase, cytochrome P450 enzyme, desaturase, or hydroxylase. Any enzyme that catalyzes a reaction step or steps in lipid synthesis, catabolism, or modification, including carrier proteins, is called a “lipid pathway enzyme”.
The term “lysate” refers to the liquid containing a mixture and/or a solution of cell contents that result from cell lysis. In some embodiments, the methods of the present invention comprise a purification of hydrocarbons or mixture of hydrocarbons in a cellular lysate. In some embodiments, the methods of the present invention comprise a purification of lipids and/or hydrocarbons and/or a mixture of hydrocarbons in a cellular lysate.
The term “lysis” refers to the rupture of the plasma membrane and if present the cell wall of a cell such that a significant amount of intracellular material escapes to the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal, or viral means. In some embodiments, the methods of the present invention comprise performing a lysis of cells or microorganisms described herein in order to separate a hydrocarbon or mixture of hydrocarbons from the contents of a bioreactor. In some embodiments, the methods of the present invention comprise performing a lysis of cells or microorganisms described herein in order to separate a lipid or mixture of lipids from the contents of a bioreactor.
The terms “microorganism” and “microbe” mean microscopic single celled life forms.
The term “molecule” means any distinct or distinguishable structural unit of matter comprising one or more atoms, and includes for example hydrocarbons, lipids, polypeptides and polynucleotides.
The term “oleaginous” refers to something that is rich in oil or produces oil in high quantities.
The term “organic compound” refers to any gaseous, liquid, or solid chemical compounds which contain carbon atoms with the following exceptions that are consider inorganic: carbides, carbonates, simple oxides of carbon, cyanides, and allotropes of pure carbon such as diamond and graphite.
“Promoter” is a control DNA sequence that regulates transcription. For purposes of the invention, a promoter may includes nucleic acid sequences near the start site of transcription that are required for proper function of the promoter, as for example, a TATA element for a promoter of polymerase II type. Promoters of the present invention can include distal enhancer or repressor elements that may lie in positions up to many thousands of base pairs away from the start site of transcription. The term “inducible promoter” refers to an operable linkage between a promoter and a nucleic acid where the promoter's mediation of nucleic acid transcription is sensitive to a specific stimulus. In some embodiments, the inducible promoter requires a cofactor which can be added to the environment of the composition comprising the nucleic acid sequence that contains the inducible promoter. An “operable linkage” refers to an operative connection between nucleic acid sequences, such as for example between a control sequence (e.g. a promoter) and another sequence that codes for a protein i.e. a coding sequence. If a promoter can regulate transcription of an exogenous gene then it is in operable linkage with the gene.
The term “syngas” (from synthetic gas or synthesis gas) refers to a gas mixture that contains various proportions of hydrogen, carbon monoxide, and carbon dioxide, and which typically also includes a variety of impurities such as methane, hydrogen sulfide, condensable gases, and tars. “Producer gas” is a related term that generally refers to gas mixes similar to syngas except for the presence of a large N2 component that results from using air directly in the gasification process.
Bacterial Species
The invention relates to chemotrophic bacterial strains that comprise one or more exogenous nucleic acid sequences. The present invention results from the discovery that chemotrophic bacteria and particular related microorganisms provide unforeseen advantages in the economic and large scale production of chemicals, oils, fuels, and other hydrocarbon or lipid substances from gaseous and waste carbon feedstocks, and also from the discovery of genetic techniques and systems for modifying these microorganisms for improved performance in these applications. The lipids and other biochemicals synthesized by the microorganisms of the present invention can be applied to uses including but not limited to petrochemical substitutes, monomers, feedstock for the production of polymers, lubricants, as ingredients in animal feed, food, personal care, and cosmetic products. In some embodiments of the present invention enzymatic and chemical processes can be utilized to produce alkenes, alkynes, hydroxy acids, diacids, and unsaturated fatty acids. Some embodiments enable the production of renewable hydrocarbons. In addition, the present invention gives methods for culturing and/or modifying chemotrophic bacteria for improved lipid yield and/or lower production costs. In some embodiments the genetically modified bacteria produce more of a certain type or types of lipid molecules as compared to the same bacteria that is not genetically modified.
The present invention relates to compositions comprising and methods of using genetically modified microorganisms to produce and/or secrete carbon-based products from conversion of gaseous carbon feedstocks including but not limited to syngas or producer gas. The present invention relates to methods and mechanisms to confer production and/or secretion of carbon-based products of interest including but not limited to ethylene, chemicals, monomers, polymers, alkenes, alkynes, hydroxy acids, diacids, unsaturated fatty acids, hydrocarbons, isoprenoids, proteins, polysaccharides, nutraceutical or pharmaceutical products or intermediates thereof in obligate or facultative chemotrophic organisms such that these organisms convert carbon dioxide and/or other forms of inorganic carbon and/or syngas and/or other C1 compounds such as methanol and/or the liquid, gaseous, and solid products of pyrolytic reactions such as pyrolysis oil, into carbon-based products of interest, and in particular the use of such organisms for the commercial production of ethylene, chemicals, monomers, polymers, alkenes, alkynes, hydroxy acids, diacids, unsaturated fatty acids, hydrocarbons, isoprenoids, proteins, polysaccharides, nutraceutical or pharmaceutical products or intermediates thereof.
Chemoautotrophs are capable of performing chemosynthetic reactions that fix CO2, and/or other forms of inorganic carbon, to organic compounds, using the potential energy stored in inorganic chemicals to drive the reaction, rather than radiant energy from light as in microorganisms performing photosynthesis [Shively et al, 1998; Smith et al, 1967; Hugler et al, 2005; Hugker et al., 2005; Scott and Cavanaugh, 2007]. Carbon fixing biochemical pathways that occur in chemoautotrophs include the reductive tricarboxylic acid cycle, the Calvin-Benson-Bassham cycle [Jessup Shively, Geertje van Kaulen, Wim Meijer, Annu Rev. Microbiol., 1998, 191-230], and the Wood-Ljungdahl pathway [Ljungdahl, 1986; Gottschalk, 1989; Lee, 2008; Fischer, 2008].
The invention relates to compositions comprising and methods of using chemoautotrophic metabolism to produce ATP for the support of ATP consuming synthetic reactions and cellular maintenance, without the co-production of methane or short chain organic acids such as acetic or butyric acid, by means of energy conserving reactions for the production of ATP using inorganic electron donors, including but not limited to the oxyhydrogen reaction.
The production of hydrocarbons or other lipids with carbon chain lengths longer than C4 is most commonly and efficiently accomplished biologically through fatty acid biosynthesis [Fischer, Klein-Marcuschamer, Stephanolpoulos, Metabolic Engineering (2008) 10, 295-304]. The initial molecule entering into the fatty acid biosynthesis pathway is acetyl-coenzyme A (acetyl-CoA), a central metabolite from which many high value biochemicals can be derived. In some embodiments, the invention utilizes microorganisms with a naturally occurring pathway for the conversion of CO, CO.sub.2 and/or H.sub.2 to acetyl-CoA. In some embodiments, the invention utilizes microorganisms that can fix CO and/or CO.sub.2 through the reductive tricarboxylic acid cycle, the Calvin-Benson-Bassham cycle, and/or the Wood-Ljungdahl pathway. In some embodiments the invention utilizes microorganisms the fix C1 compounds through a methanotropic pathway. In some embodiments the microorganisms naturally produce enzymes that catalyze the fixation of gaseous inorganic carbon to produce acetyl-CoA, utilizing gaseous electron donors such as are present in syngas as reducing agents, with such enzymatic proteins including but not limited to acetyl-CoA synthase, acetyl-CoA synthase disulfide reductase, cobalamide corrinoid/iron-sulfur protein, carbon monoxide dehydrogenase, hydrogenase, and methyltransferase.
Unlike methanogenic, acetogenic and solventogenic pathways, present in methanogens and acetogens respectively, which can produce short chain organic compounds (C1-C4) with net ATP production or zero net consumption, fatty acid synthesis involves net ATP consumption. For example the following gives the net reaction for synthesis of Palmitic acid (C16) starting from Acetyl-CoA:

8Acetyl−CoA+7ATP+H2O+14NADPH+14H+−>Palmitic acid+8CoA+14NADP++7ADP+7Pi

A drawback with using an obligate methanogen or acetogen in a GTL process for the production of lipids, is the obligate use of CO2 as an electron acceptor for the production of ATP that is needed for fatty acid synthesis. If H2 is the electron donor, the ATP produced per H2 consumed in an acetogen or methanogen is relatively low: one ATP per 4H2 for methane [Thauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W. & Hedderich, R. Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Microbiol 6, 579-591, doi:nrmicro1931 [pii]] or acetic acid production, and one ATP per 10H2 for butyric acid production [Papoutsakis, Biotechnology & Bioengineering (1984) 26, 174-187; Heise, Muller, Gottschalk, J. of Bacteriology (1989) 5473-5478; Lee, Park, Jong, Nielsen, Kim, Jung, Biotechnology & Bioengineering (2008) 101, 2, 209-228]. In some embodiments, the invention relates to a microorganism or compositions comprising a microorganism, wherein the microorganism produces ATP from an inorganic electron donor such as but not limited to H2 without synthesis of methane or short chain organic acids.
Hydrogen-oxidizing microorganisms that use more electronegative electron acceptors in energy conserving reactions for ATP production, such as but not limited to hydrogenotrophic oxyhydrogen or knallgas microbes that link the oxyhydrogen reaction, 2 H2+O2−>2 H2 O, to ATP production, can produce more ATP per H2 consumed than acetogens or methanogens. For example knallgas microorganisms can produce up to two ATP per H2 consumed [Bongers, J. Bacteriology, (October 1970) 145-151], which is eight times more ATP produced per H2 consumed than what can be produced in microorganisms undergoing methanogenesis or acetogenesis. For this reason using microorganisms that can utilize more electronegative electron acceptors in the production of ATP, such as but not limited to knallgas microbes, in fatty acid biosynthesis from syngas or H2, can be more efficient for supporting fatty acid biosynthesis than using the acetogens or methanogens that are currently used in biological GTL technologies. In some embodiments, the invention relates to a microorganism or compositions comprising a microorganism, wherein the microorganism is a knallgas microbe and comprises at least one or more exogenous nucleic acid sequences that encodes one or more enzymes to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest including but not limited to ethylene, chemicals, monomers, polymers, alkenes, alkynes, hydroxy acids, diacids, unsaturated fatty acids, hydrocarbons, isoprenoids, proteins, polysaccharides, nutraceutical or pharmaceutical products or intermediates thereof. The invention relates to a genetically modified microorganism and compositions comprising such a microorganism, wherein the microorganism comprises one or more exogenous genes and wherein the microorganism grows on carbon-containing gas or utilizes a gaseous feedstock selected from syngas, CO2, H2, CO, or mixtures of gas comprising one or more gases selected from syngas, CO2, H2, or CO.
The invention relates to a cell and compositions comprising a cell of the class Actinobacteria comprising at least one exogenous gene. The invention also relates to cells and compositions comprising cells of the family of Nocardiaceae comprising at least one exogenous gene. The invention relates to cells and compositions comprising cells of Corynebacterium, Gordonia, Rhodococcus, Mycobacterium and Tsukamurella comprising at least one exogenous gene. In some embodiments, the invention relate to cells of the family of Nocardiaceae comprising an exogenous gene, wherein the cell is not a cell of the genus Mycobacterium. In some embodiments, the invention provides a cell and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, and in some embodiments the cell is a strain of the species Rhodococcus sp., Rhodococcus opacus, Rhodococcus aurantiacus; Rhodococcus baikonurensis; Rhodococcus boritolerans; Rhodococcus equi; Rhodococcus coprophilus; Rhodococcus corynebacterioides; Nocardia corynebacterioides (synonym: Nocardia corynebacterioides); Rhodococcus erythropolis; Rhodococcus fascians; Rhodococcus globerulus; Rhodococcus gordoniae; Rhodococcus jostii Rhodococcus koreensis; Rhodococcus kroppenstedtii; Rhodococcus maanshanensis; Rhodococcus marinonascens; Rhodococcus opacus; Rhodococcus percolatus; Rhodococcus phenolicus; Rhodococcus polyvorum; Rhodococcus pyridinivorans; Rhodococcus rhodochrous; Rhodococcus rhodnii; (synonym: Nocardia rhodnii); Rhodococcus ruber (synonym: Streptothrix rubra); Rhodococcus sp. RHA1; Rhodococcus triatomae; Rhodococcus tukisamuensis; Rhodococcus wratislaviensis (synonym: Tsukamurella wratislaviensis); Rhodococcus yunnanensis; Rhodococcus zopfii. In some embodiments the cell comprising one or more exogenous genes is strain Rhodococcus opacus DSM number 43205 or 43206. In some embodiments the cell comprising one or more exogenous genes is strain Rhodococcus sp. DSM number 3346. In some embodiments, the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the cell or composition comprising a cell of Rhodococcus is non-infectious to animals and/or plants. In some embodiments, the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the Rhodococcus cell or composition comprising a Rhodococcus cell is non-infectious to humans. In some embodiments, the invention provides cells and compositions comprising a cell of the genus Rhodococcus comprising an exogenous gene, wherein the Rhodococcus cell or composition comprising a Rhodococcus cell is non-infectious to plants. In some embodiments, the invention provides cells and compositions comprising cells of the genus Rhodococcus comprising an exogenous gene, wherein, if the cell is from Rhodococcus equi or Rhodococcus fascians species, the species is non-infectious to animals and/or plants. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is not a species selected from Rhodococcus equi or Rhodococcus fascians. 
In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing any acrylic acid or acrylamide. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell produces less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in acrylamide or acrylic/methylacrylic acid. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is not from the species Rhodococcus rhodochrous. In some embodiments, the invention relates to Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing 10-hydroxy-12-octadecenoic acid. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is unable to produce more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in 10-hydroxy-12-octadecenoic acid. In some embodiments, the invention relates to Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is incapable of producing optically-active 4-amino-3-hydroxybutyric acid. In some embodiments, the invention relates to a Rhodococcus cell or composition comprising a Rhodococcus cell, wherein the cell is unable to produce more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of its weight of total dry cellular matter in optically-active 4-amino-3-hydroxybutyric acid.
In some embodiments, the cell or compositions comprising one of more cells is not E. coli. In some embodiments, the cell or compositions comprising one of more cells is from the genus Rhodococcus but is not for the species equi. In some embodiments, the cell of the present invention is not pathogenic to animals or plants. In some embodiments, the cell of the present invention is not pathogenic to humans. In some embodiments, the cell or compositions comprising one of more cells is from the genus Ralstonia. In some embodiments, the cell or compositions comprising one of more cells is from the species Ralstonia eutropha. In some embodiments the cell comprising one or more exogenous genes is strain Cupriavidus necator DSM number 531 or 541.
In some embodiments, the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 50, 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 11 or 12. In some embodiments, the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 11. In some embodiments, the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 12. In some embodiments, the cell or compositions comprising the one or more cells have a 16S rRNA sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% nucleotide homology to one or more of SEQ ID NOs: 13.
In some embodiments the microorganism of the claimed invention is not dependent upon light to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism of the claimed invention does not require any type of sugar to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism of the claimed invention does not require any type of organic compound to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism of the claimed invention does not require any type of fixed carbon to grow and/or metabolize and/or synthesize lipid molecules. In some embodiments, the microorganism can grow and/or metabolize lipids in a slightly anaerobic or extremely anaerobic environment. In some embodiments, the microorganism of the claimed invention is a facultative microorganism
Microbial culturing in the present invention is performed both for the sake of implementing genetic modifications, and for production of organic compounds, and specifically lipids and/or hydrocarbons (e.g., alkenes, alkynes, unsaturated fatty acids, or triacylglycerols, hydroxy acids, diacids). Microbial culturing with the aim of genetic manipulation is generally performed at a small benchtop scale and often under conditions that select for genetically modified traits. Microbial culturing aimed at the commercial production of organic compounds and specifically lipids and/or hydrocarbons is typically performed in bioreactors at much greater scale (e.g., 500 L, 1,000 L 5,000 L, 10,000 L, 50,000 L, 100,000 L, 1,000,000 L bioreactor volumes and higher). In certain embodiments the chemoautotrophs of the present invention are grown in a liquid media inside a bioreactor using the methods of the invention. In some embodiments, the bioreactor containing the microorganisms is constructed of opaque materials that keep the culture in darkness. Bioreactors constructed out of opaque materials such as steel or reinforced concrete can be designed to have extremely big working volumes. In some embodiments of the present invention steel fermenters 50,000 liter and greater in volume are utilized. In some embodiments of the present invention egg-shape or cylindrical digesters 3,000,000 liters and greater in volume are utilized. In some embodiments, the bioreactor comprising the microorganism does not allow light to penetrate its interior.
The bioreactor or fermentor is used to culture cells through the various phases of their physiological cycle. A bioreactor is utilized for the cultivation of cells, which may be maintained at particular phases in their growth curve. The use of bioreactors is advantageous in many ways for cultivating chemoautotrophic growth. For certain embodiments, oleaginous cell mass, which is used to produce oleochemicals, is grown to high densities in liquid suspension. Generally the control of growth conditions including control of dissolved carbon dioxide, oxygen, and other gases such as hydrogen, as well as other dissolved nutrients, trace elements, temperature and pH, is facilitated in a bioreactor.
Nutrient media as well as gases can be added to the bioreactor as either a batch addition, or periodically, or in response to a detected depletion or programmed set point, or continuously over the period the culture is grown and/or maintained. For certain embodiments, the bioreactor at inoculation is filled with a starting batch of nutrient media and/or gases at the beginning of growth, and no additional nutrient media and/or gases are added after inoculation. For certain embodiments, nutrient media and/or gases are added periodically after inoculation. For certain embodiments, nutrient media and/or gas is added after inoculation in response to a detected depletion of nutrient and/or gas. For certain embodiments, nutrient media and/or gas is added continuously after inoculation.
For certain embodiments the bioreactors have mechanisms to enable mixing of the nutrient media that include but are not limited to spinning stir bars, blades, impellers, or turbines, spinning, rocking, or turning vessels, gas lifts and sparging. The culture media may be mixed continuously or intermittently.
The ports that are standard in bioreactors may be utilized to deliver, or withdraw, gases, liquids, solids, and/or slurries, into the bioreactor vessel enclosing the microbes of the present invention. Many bioreactors have multiple ports for different purposes (e.g. ports for media addition, gas addition, probes for pH and DO, sampling), and a given port may be used for various purposes during the course of a fermentation run. As an example, a port might be used to add nutrient media to the bioreactor at one point in time and at another time might be used for sampling. Preferably, the multiple use of a sampling port can be performed without introducing contamination or invasive species into the growth environment. A valve or other actuator enabling control of the sample flow or continuous sampling can be provided to a sampling port. For certain embodiments the bioreactors are equipped with at least one port suitable for culture inoculation that can additionally serve other uses including the addition of media or gas. Bioreactors ports enable control of the gas composition and flow rate into the culture environment. For example the ports can be used as gas inlets into the bioreactor through which gases are pumped. For some embodiments gases that may be pumped into a bioreactor include syngas, producer gas, hydrogen gas, CO2, air, air/CO2 mixtures, ammonia, nitrogen, noble gases, such as argon, as well as other gases. In some embodiments that CO.sub.2 may come from sources including but are not limited to: CO.sub.2 from the gasification of organic matter; CO.sub.2 from the calcination of limestone, CaCO.sub.3, to produce quicklime, CaO; CO.sub.2 from methane steam reforming, such as the CO.sub.2 byproduct from ammonia or hydrogen production; combustion; CO.sub.2 byproduct of sugar fermentation; CO.sub.2 byproduct from sodium phosphate production; geologically or geothermally produced CO.sub.2. Raising the gas flow rate into a bioreactor can enhance mixing of the culture and produce turbulence if the gas inlet is positioned under the surface of the liquid media such that gas bubbles or sparges up through the media. In some embodiments, a bioreactor comprises gas outlet ports for gas escape and pressure release. In some embodiments, gas inlets and outlets are preferably equipped with check valves to prevent gas backflow.
The present invention relates to bioreactors that comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme. The present invention relates to a system of at least one bioreactor that comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme. In some embodiments, the system comprises two or more, three or more, or four or more bioreactors, at least one of which comprise a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme. In some embodiments, the system of bioreactors comprises at least a first and second bioreactor, wherein the first bioreactor comprises a cell, which comprises at least one exogenous nucleic acid sequences that encodes a lipid pathway enzyme; and wherein the second bioreactor comprises a microorganism derived from a different species, wherein the microorganism from a different species comprises at least one exogenous nucleic acid sequence that encodes a lipid pathway enzyme. In some embodiments, the system of bioreactors comprises a first bioreactor that comprises the cell of the present invention and a second bioreactor comprising a microalgal, yeast, or bacterial cell.
In some embodiments, the cells of the present invention are capable of producing desaturated alkanes between 8 and 18 carbon atoms long at greater than 18 grams per liter volume of culture per three day period. In some embodiments, the cells of the present invention are capable of producing desaturated hydrocarbons between 8 and 18 carbon atoms long at greater than or equal to 18 grams per liter volume of culture per three day period, wherein the desatruated hydrocarbons are desatuated at a carbon position other than carbon-9.
Genetic Modifications
The present invention relates to methods of modifying a bacterial cell to express one or more exogenous nucleic acid sequences that encodes one or more enzymes to enable fixation of a carbon-containing gas feedstock into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequences. Methods of selecting and manufacturing nucleic acid sequences for modification of bacterial cells are known and can be performed by transformation, electroporation, phage infection of bacteria, or other techniques for nucleic acid transfer generally known in the art. Standard recombinant DNA and molecular cloning techniques useful for the invention are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987), all of which are incorporated by reference in their entireties.
The invention relates to genetic constructs comprising one or more exogenous genes that encode one or more amino acid sequences to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequence or sequences. Another aspect of the present invention relates to compositions that comprise at least one bacterial cell, which comprises at least one nucleic acid sequence that encodes at least one exogenous amino acid sequence that functions as a fatty acid acyl-ACP reductase, a fatty acid aldehyde decarbonylase and/or a thioesterase. In some embodiments, the bacterial cell is transformed with one or more, two or more, three or more, four or more, or five or more exogenous nucleic acid sequences that encode one or more amino acid sequences to enable fixation of a carbon-containing gas feedstock, including but not limited to syngas or producer gas, into useful carbon-based products of interest in an amount greater than an amount of carbon-based products produced by the same bacterial cell that does not express the exogenous nucleic acid sequence or sequences. According to the present invention, genetic material that encodes the enzyme is delivered to a bacterial cell in an expressible form. The genetic material, DNA or RNA, is taken up by the cells of the invention and expressed. The enzyme or enzymes that are thereby produced can biochemically modify lipid molecules to remove or add hydroxyl groups, remove or add carbonyl groups, remove or add carbon-carbon double bonds, remove or add carbon-carbon triple bonds, remove or add aldehyde groups, remove or add hydroxy groups, remove or add carboxylic acid groups, or remove or add ester groups to lipid molecules in lipid.
In some embodiments, the genetic constructs of the present invention comprise DNA, RNA, or combinations of both DNA and RNA. In some embodiments, the genetic construct of the present invention is a plasmid. It will be appreciated that, in some embodiments, the plasmid contains a variety of open reading frames (ORFs) encoding proteins of many diverse functions, including those enzymes that enable hydrocarbon or lipid modification, glutathione-S transferase (GST) activity, origins of replication, multiple cloning sites, promoters, and/or termination sequences. It is contemplated therefore that a host cell transformed with the plasmid will demonstrate the ability to modify a variety of lipids as well as maintain its copy number in the cytoplasm of the cell. The glutathione-S transferases (GSTs) represent a large group of detoxification enzymes. GSTs catalyze the conjugation of glutathione, homoglutathione and other glutathione-like analog via sulfhydryl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds. GST genes are found in both prokaryotic (e.g., E. coli) and eukaryotic organisms (e.g., yeast, plant and human). Although the homologies between the GSTs from prokaryotes and eukaryotes were low, many of the residues assigned to be important for the enzymatic function or structure in the eukaryotes were found to be conserved in prokaryotic GSTs (Nishida et al., J. Biol Chem 269:32536-32541 (1994)). It has been suggested that bacterial GST may represent a defense against the effects of antibiotics (Piccolomini et al., J Gen Microbiol 135:3119-3125 (1989)). Accordingly it is contemplated that a host strain transformed with the plasmid will have the ability detoxify harmful compounds via conjugation of those compounds to glutathione.
In some embodiments, the instant plasmid additionally encodes a variety of maintenance proteins, useful for maintaining, stabilizing and replicating the plasmid. It is contemplated that these genes may be used in conjunction with other bacterial plasmids deficient in these functions for the increased stabilization or robust maintenance of the plasmid. In some embodiments, the plasmid comprises maintenance proteins of particular interest including the REP origin of replication (encoded by ORF 38) the TRA proteins (TRAI, TRAJ and TRAK, encoded by ORF's 23, 24 and 25 respectively) and the VAG proteins (VAGD and VAGC, encoded by ORF's 33 and 34 respectively). The tra gene family is known to be involved in plasmid conjugation, a process that promotes DNA transfer from a donor to a recipient cell mediated by physical contact (Firth et al, Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM press (1996)). Among tra gene products, TraI and TraK proteins are reported to be required for efficient plasmid site-specific recombination (Paterson et al. J. Bacteriol 181:2572-2583 (1999)). Furthermore, TraI is required for conjugal DNA transfer. Fukuda and Ohtsubo (Genes Cells 2:735-751 (1997)) reported that TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. TraJ, traJ gene product, regulates transcription originating at the tra operon promoter P.sub.traY. (Firth et al., Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM press (1996)). The stabilization proteins VAGC and VAGD encoded by vagC and vagD are involved in the maintaining the plasmid as an autonomous replicating unit. Bacterial maintenance proteins of particular interest on the pSeq and pVer plasmids include.







SEQ ID: 01


TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT


 


GCAGCTCCCG GAGACGGTCA CAGCTTGTCT GTAAGCGGAT


 


GCCGGGAGCA GACAAGCCCG AGCGCGCAAA GCCACTACTG


 


CCACTTTTGG AGACTGTGTA CGTCGAGGGC CTCTGCCAGT


 


GTCGAACAGA CATTCGCCTA CGGCCCTCGT CTGTTCGGGC


 


TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG


 


CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC


 


ACCATATGCG GTGTGAAATA AGTCCCGCGC AGTCGCCCAC


 


AACCGCCCAC AGCCCCGACC GAATTGATAC GCCGTAGTCT


 


CGTCTAACAT GACTCTCACG TGGTATACGC CACACTTTAT


 


CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC


 


ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC


 


GGTGCGGGCC TCTTCGCTAT GGCGTGTCTA CGCATTCCTC


 


TTTTATGGCG TAGTCCGCGG TAAGCGGTAA GTCCGACGCG


 


TTGACAACCC TTCCCGCTAG CCACGCCCGG AGAAGCGATA


 


TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT


 


AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT


 


AAAACGACGG CCAGTGCCAA ATGCGGTCGA CCGCTTTCCC


 


CCTACACGAC GTTCCGCTAA TTCAACCCAT TGCGGTCCCA


 


AAAGGGTCAG TGCTGCAACA TTTTGCTGCC GGTCACGGTT


 


GCTTGCATGC CTGCAGGTCG ACGGGCCCGG GATCCGATGC


 


TCTTCCGCTA AGATCTGCCG CGGCCGCGTC CTCAGAAGAA


 


CTCGTCAAGA AGGCGATAGA CGAACGTACG GACGTCCAGC


 


TGCCCGGGCC CTAGGCTACG AGAAGGCGAT TCTAGACGGC


 


GCCGGCGCAG GAGTCTTCTT GAGCAGTTCT TCCGCTATCT


 


AGGCGATGCG CTGCGAATCG GGAGCGGCGA TACCGTAAAG


 


CACGAGGAAG CGGTCAGCCC ATTCGCCGCC AAGCTCTTCA


 


GCAATATCAC GGGTAGCCAA TCCGCTACGC GACGCTTAGC


 


CCTCGCCGCT ATGGCATTTC GTGCTCCTTC GCCAGTCGGG


 


TAAGCGGCGG TTCGAGAAGT CGTTATAGTG CCCATCGGTT


 


CGCTATGTCC TGATAGCGGT CCGCCACACC CAGCCGGCCA


 


CAGTCGATGA ATCCAGAAAA GCGGCCATTT TCCACCATGA


 


TATTCGGCAA GCAGGCATCG GCGATACAGG ACTATCGCCA


 


GGCGGTGTGG GTCGGCCGGT GTCAGCTACT TAGGTCTTTT


 


CGCCGGTAAA AGGTGGTACT ATAAGCCGTT CGTCCGTAGC


 


CCATGGGTCA CGACGAGATC CTCGCCGTCG GGCATGCGCG


 


CCTTGAGCCT GGCGAACAGT TCGGCTGGCG CGAGCCCCTG


 


ATGCTCTTCG TCCAGATCAT GGTACCCAGT GCTGCTCTAG


 


GAGCGGCAGC CCGTACGCGC GGAACTCGGA CCGCTTGTCA


 


AGCCGACCGC GCTCGGGGAC TACGAGAAGC AGGTCTAGTA


 


CCTGATCGAC AAGACCGGCT TCCATCCGAG TACGTGCTCG


 


CTCGATGCGA TGTTTCGCTT GGTGGTCGAA TGGGCAGGTA


 


GCCGGATCAA GCGTATGCAG GGACTAGCTG TTCTGGCCGA


 


AGGTAGGCTC ATGCACGAGC GAGCTACGCT ACAAAGCGAA


 


CCACCAGCTT ACCCGTCCAT CGGCCTAGTT CGCATACGTC


 


CCGCCGCATT GCATCAGCCA TGATGGATAC TTTCTCGGCA


 


GGAGCAAGGT GGGATGACAG GAGATCCTGC CCCGGCACTT


 


CGCCCAATAG CAGCCAGTCC GGCGGCGTAA CGTAGTCGGT


 


ACTACCTATG AAAGAGCCGT CCTCGTTCCA CCCTACTGTC


 


CTCTAGGACG GGGCCGTGAA GCGGGTTATC GTCGGTCAGG


 


CTTCCCGCTT CAGTGACAAC GTCGAGCACA GCTGCGCAAG


 


GAACGCCCGT CGTGGCCAGC CACGATAGCC GCGCTGCCTC


 


GTCCTGCAGT TCATTCAGGG GAAGGGCGAA GTCACTGTTG


 


CAGCTCGTGT CGACGCGTTC CTTGCGGGCA GCACCGGTCG


 


GTGCTATCGG CGCGACGGAG CAGGACGTCA AGTAAGTCCC


 


CACCGGACAG GTCGGTCTTG ACAAAAAGAA CCGGGCGCCC


 


CTGCGCTGAC AGCCGGAACA CGGCGGCATC AGAGCAGCCG


 


ATTGTCTGTT GTGCCCAGTC GTGGCCTGTC CAGCCAGAAC


 


TGTTTTTCTT GGCCCGCGGG GACGCGACTG TCGGCCTTGT


 


GCCGCCGTAG TCTCGTCGGC TAACAGACAA CACGGGTCAG


 


ATAGCCGAAT AGCCTCTCCA CCCAAGCGGC CGGAGAACCT


 


GCGTGCAATC CATCTTGTTC AATCATGATA TCCCTTAATT


 


AACCGTTAAC ACTAGTTCAG TATCGGCTTA TCGGAGAGGT


 


GGGTTCGCCG GCCTCTTGGA CGCACGTTAG GTAGAACAAG


 


TTAGTACTAT AGGGAATTAA TTGGCAATTG TGATCAAGTC


 


TCCATCTCGC CGTGTATGCG GGCCTGACGG ATCAACGTTC


 


CCACCGAGCC AGTCGAGATG TTCATCTGGT CGGCGATCTG


 


CCGGTACTTC AAACCTTGTT AGGTAGAGCG GCACATACGC


 


CCGGACTGCC TAGTTGCAAG GGTGGCTCGG TCAGCTCTAC


 


AAGTAGACCA GCCGCTAGAC GGCCATGAAG TTTGGAACAA


 


TGCGCAGTTC CACAGCCTTC TTGCGGCGTT CCTGCGCACG


 


AGCGATGTAG TCGCCTCGGT CTTCGGCGAC GAGCCGTTTG


 


ATGGTGCTTT TCGAGACGCC ACGCGTCAAG GTGTCGGAAG


 


AACGCCGCAA GGACGCGTGC TCGCTACATC AGCGGAGCCA


 


GAAGCCGCTG CTCGGCAAAC TACCACGAAA AGCTCTGCGG


 


GAACTTGTCA GCCAACTCCT GCGCGGTCTG CGTGCGACGC


 


ATCACGCGTT CTGCAGCACC CATCAGTCCG TCCCCTCTGC


 


TGCTGCGAAC AGTGCCGATC CTTGAACAGT CGGTTGAGGA


 


CGCGCCAGAC GCACGCTGCG TAGTGCGCAA GACGTCGTGG


 


GTAGTCAGGC AGGGGAGACG ACGACGCTTG TCACGGCTAG


 


GATCGACCTT CTTGAGCTTC GGCCGCGGCG CGGTGGCGTT


 


CTTCCGTACC GCTTCCGTTT TTGCGCTGCT GCTCACTTTG


 


CCGCGGCGTG CCTGGATTTT CTAGCTGGAA GAACTCGAAG


 


CCGGCGCCGC GCCACCGCAA GAAGGCATGG CGAAGGCAAA


 


AACGCGACGA CGAGTGAAAC GGCGCCGCAC GGACCTAAAA


 


CGAGAACTCG GCGGCGGTGA AGGTGCGGTG GGTCCAGTGG


 


GCGACTGATT TGCCGATCTG CTCGGCCTCG GCCCGACTCA


 


TGGGGCCGAT CCCGTCGTTG GCTCTTGAGC CGCCGCCACT


 


TCCACGCCAC CCAGGTCACC CGCTGACTAA ACGGCTAGAC


 


GAGCCGGAGC CGGGCTGAGT ACCCCGGCTA GGGCAGCAAC


 


GCGTCGAGGG TGAAGTTGGT CAGGGCGGTG AAGTCGGTGA


 


CCATCTGCCG CCACACAGTG ATCGACGGGT AGTTCTGTTT


 


CCGGATCTCG CGGTAGGCCC CGCAGCTCCC ACTTCAACCA


 


GTCCCGCCAC TTCAGCCACT GGTAGACGGC GGTGTGTCAC


 


TAGCTGCCCA TCAAGACAAA GGCCTAGAGC GCCATCCGGG


 


ATTCCCGGGT GCGGTCGAAC AGTTCGACGT TCCGGCCCGT


 


TTCGGTCCTG ACCTGTGTCT TGCGGCCGTA GTCCGGTGGG


 


GCGGGGAAAC GGTCACCGAG TAAGGGCCCA CGCCAGCTTG


 


TCAAGCTGCA AGGCCGGGCA AAGCCAGGAC TGGACACAGA


 


ACGCCGGCAT CAGGCCACCC CGCCCCTTTG CCAGTGGCTC


 


CGCTTTTGCG AGGCCTTTGA GCGAGTACGG ATCCGAGGGA


 


CCCCAGACCG TCGTCCAGTG CGGGTGGATC GGGTTCTGGG


 


TGAGCTGCTG CGCGTAGCCC GCGAAAACGC TCCGGAAACT


 


CGCTCATGCC TAGGCTCCCT GGGGTCTGGC AGCAGGTCAC


 


GCCCACCTAG CCCAAGACCC ACTCGACGAC GCGCATCGGG


 


TGATCGGCGC CGACCACCGA GGCGATCAGC CCCTGGTTCA


 


CCCGGTCGTA GAGCCGCAGC GGGCCCTGTC GGGCTGCCTG


 


GAGGGTGTAG ACCGGGCTTT ACTAGCCGCG GCTGGTGGCT


 


CCGCTAGTCG GGGACCAAGT GGGCCAGCAT CTCGGCGTCG


 


CCCGGGACAG CCCGACGGAC CTCCCACATC TGGCCCGAAA


 


CGAGCAGCCA CCACAGGTGC GCGTGCTCGG TCGCGGGATT


 


GATCGTCATC ACGGTCGGAT CGGGCAGATC CGCGTTACGT


 


GCGGCCCACT GCGCCTGGTC GCTCGTCGGT GGTGTCCACG


 


CGCACGAGCC AGCGCCCTAA CTAGCAGTAG TGCCAGCCTA


 


GCCCGTCTAG GCGCAATGCA CGCCGGGTGA CGCGGACCAG


 


GTCGTCCACG TCGAGCACCA AGCCCAACCT GATCGACGGG


 


GTGCGGGCCG CAATGTAGCG GCGGGTGAGC GCCTCCGCGC


 


GCGGCTGCGG CCACTGCCCG CAGCAGGTGC AGCTCGTGGT


 


TCGGGTTGGA CTAGCTGCCC CACGCCCGGC GTTACATCGC


 


CGCCCACTCG CGGAGGCGCG CGCCGACGCC GGTGACGGGC


 


TCCCGGACGT AGTCATCCGT CGCGTGCGGG TATTTGAACC


 


GCCAGCGGTC CAACCAGGCG TCAACAGCAG CGGTCATGAC


 


CGCCAAGCTA GGGCCGGATC AGGGCCTGCA TCAGTAGGCA


 


GCGCACGCCC ATAAACTTGG CGGTCGCCAG GTTGGTCCGC


 


AGTTGTCGTC GCCAGTACTG GCGGTTCGAT CCCGGCCTAG


 


TGTACCGATC GGGGGAGGCG CGCCGCAAAT TATTTAAGAG


 


TCTCGCTAGC AAACCATGTC AGGTGTTGCG GTGGGTTCCG


 


GGTAAACCTC CACCCGAATT ACATGGCTAG CCCCCTCCGC


 


GCGGCGTTTA ATAAATTCTC AGAGCGATCG TTTGGTACAG


 


TCCACAACGC CACCCAAGGC CCATTTGGAG GTGGGCTTAA


 


ATTTAAGAGT CTCGCTAGCT AAGCCCTATC TGATGCTGCG


 


CGGGGGGTCC TTCGCACTGA ATCTCAAAGG TGGCCGGCTG


 


AATTTCGTCG CGCGAAAACC TAAATTCTCA GAGCGATCGA


 


TTCGGGATAG ACTACGACGC GCCCCCCAGG AAGCGTGACT


 


TAGAGTTTCC ACCGGCCGAC TTAAAGCAGC GCGCTTTTGG


 


TCCCTGGACA GTTCTGGAAT TCAGCAAGAG GTGTGTCTGA


 


ACTTCGGTGT TTTTTTGGGG GGTGACTCCA GCGGGGTGGG


 


CACAACGCGA ACAGAGACCT AGGGACCTGT CAAGACCTTA


 


AGTCGTTCTC CACACAGACT TGAAGCCACA AAAAAACCCC


 


CCACTGAGGT CGCCCCACCC GTGTTGCGCT TGTCTCTGGA


 


TGTGTGTACG ACGGCGGGAG GTAAGTCGGG TACGGCTCGG


 


ACTGCGGTAG AGCAACCGTC GAATCGATTT CGAGCAGAGC


 


GAGCAGAGCA AGATATTCCA ACACACATGC TGCCGCCCTC


 


CATTCAGCCC ATGCCGAGCC TGACGCCATC TCGTTGGCAG


 


CTTAGCTAAA GCTCGTCTCG CTCGTCTCGT TCTATAAGGT


 


AAACTCCGGG GTTCCTCGGC GGCCTCCCCC GTCTGTTTGC


 


TCAACCGAGG GAGACCTGGC GGTCCCGCGT TTCCGGACGC


 


GCGGGACCGC CTACCGCTCG TTTGAGGCCC CAAGGAGCCG


 


CCGGAGGGGG CAGACAAACG AGTTGGCTCC CTCTGGACCG


 


CCAGGGCGCA AAGGCCTGCG CGCCCTGGCG GATGGCGAGC


 


AGAGCGGAAG AGCATCTAGA TGCATTCGCG AGGTACCGAG


 


CTCGAATTCG TAATCATGGT CATAGCTGTT TCCTGTGTGA


 


AATTGTTATC CGCTCACAAT TCTCGCCTTC TCGTAGATCT


 


ACGTAAGCGC TCCATGGCTC GAGCTTAAGC ATTAGTACCA


 


GTATCGACAA AGGACACACT TTAACAATAG GCGAGTGTTA


 


TCCACACAAC ATACGAGCCG GAAGCATAAA GTGTAAAGCC


 


ATGGGGTGCC TATGAGTGAG CTAACTCACA TTAATTGCGT


 


TGCGCTCACT GCCCGCTTTC AGGTGTGTTG TATGCTCGGC


 


CTTCGTATTT CACATTTCGG ACCCCACGGA TTACTCACTC


 


GATTGAGTGT AATTAACGCA ACGCGAGTGA CGGGCGAAAG


 


CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT TAATGAATCG


 


GCCAACGCGC GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC


 


TTCCGCTTCC TCGCTCACTG GTCAGCCCTT TGGACAGCAC


 


GGTCGACGTA ATTACTTAGC CGGTTGCGCG CCCCTCTCCG


 


CCAAACGCAT AACCCGCGAG AAGGCGAAGG AGCGAGTGAC


 


ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC


 


AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA


 


GGGGATAACG CAGGAAAGAA TGAGCGACGC GAGCCAGCAA


 


GCCGACGCCG CTCGCCATAG TCGAGTGAGT TTCCGCCATT


 


ATGCCAATAG GTGTCTTAGT CCCCTATTGC GTCCTTTCTT


 


CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA


 


AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC


 


CTGACGAGCA TCACAAAAAT GTACACTCGT TTTCCGGTCG


 


TTTTCCGGTC CTTGGCATTT TTCCGGCGCA ACGACCGCAA


 


AAAGGTATCC GAGGCGGGGG GACTGCTCGT AGTGTTTTTA


 


CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT


 


AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG


 


CTCTCCTGTT CCGACCCTGC GCTGCGAGTT CAGTCTCCAC


 


CGCTTTGGGC TGTCCTGATA TTTCTATGGT CCGCAAAGGG


 


GGACCTTCGA GGGAGCACGC GAGAGGACAA GGCTGGGACG


 


CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG


 


CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT


 


TCGGTGTAGG TCGTTCGCTC GCGAATGGCC TATGGACAGG


 


CGGAAAGAGG GAAGCCCTTC GCACCGCGAA AGAGTATCGA


 


GTGCGACATC CATAGAGTCA AGCCACATCC AGCAAGCGAG


 


CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC


 


CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC


 


CGGTAAGACA CGACTTATCG GTTCGACCCG ACACACGTGC


 


TTGGGGGGCA AGTCGGGCTG GCGACGCGGA ATAGGCCATT


 


GATAGCAGAA CTCAGGTTGG GCCATTCTGT GCTGAATAGC


 


CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA


 


GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC


 


TAACTACGGC TACACTAGAA GGTGACCGTC GTCGGTGACC


 


ATTGTCCTAA TCGTCTCGCT CCATACATCC GCCACGATGT


 


CTCAAGAACT TCACCACCGG ATTGATGCCG ATGTGATCTT


 


GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC


 


CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA


 


ACCACCGCTG GTAGCGGTGG CCTGTCATAA ACCATAGACG


 


CGAGACGACT TCGGTCAATG GAAGCCTTTT TCTCAACCAT


 


CGAGAACTAG GCCGTTTGTT TGGTGGCGAC CATCGCCACC


 


TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA


 


GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG


 


ACGCTCAGTG GAACGAAAAC AAAAAAACAA ACGTTCGTCG


 


TCTAATGCGC GTCTTTTTTT CCTAGAGTTC TTCTAGGAAA


 


CTAGAAAAGA TGCCCCAGAC TGCGAGTCAC CTTGCTTTTG


 


TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA


 


TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA


 


ATCAATCTAA AGTATATATG AGTGCAATTC CCTAAAACCA


 


GTACTCTAAT AGTTTTTCCT AGAAGTGGAT CTAGGAAAAT


 


TTAATTTTTA CTTCAAAATT TAGTTAGATT TCATATATAC


 


AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA


 


GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA


 


GTTGCCTGAC TCCCCGTCGT TCATTTGAAC CAGACTGTCA


 


ATGGTTACGA ATTAGTCACT CCGTGGATAG AGTCGCTAGA


 


CAGATAAAGC AAGTAGGTAT CAACGGACTG AGGGGCAGCA


 


GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC


 


AGTGCTGCAA TGATACCGCG AGACCCACGC TCACCGGCTC


 


CAGATTTATC AGCAATAAAC CATCTATTGA TGCTATGCCC


 


TCCCGAATGG TAGACCGGGG TCACGACGTT ACTATGGCGC


 


TCTGGGTGCG AGTGGCCGAG GTCTAAATAG TCGTTATTTG


 


CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA


 


CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA


 


AGCTAGAGTA AGTAGTTCGC GTCGGTCGGC CTTCCCGGCT


 


CGCGTCTTCA CCAGGACGTT GAAATAGGCG GAGGTAGGTC


 


AGATAATTAA CAACGGCCCT TCGATCTCAT TCATCAAGCG


 


CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG


 


CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC


 


AGCTCCGGTT CCCAACGATC GTCAATTATC AAACGCGTTG


 


CAACAACGGT AACGATGTCC GTAGCACCAC AGTGCGAGCA


 


GCAAACCATA CCGAAGTAAG TCGAGGCCAA GGGTTGCTAG


 


AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG


 


GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT


 


TGGCCGCAGT GTTATCACTC TTCCGCTCAA TGTACTAGGG


 


GGTACAACAC GTTTTTTCGC CAATCGAGGA AGCCAGGAGG


 


CTAGCAACAG TCTTCATTCA ACCGGCGTCA CAATAGTGAG


 


ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC


 


CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC


 


CAAGTCATTC TGAGAATAGT TACCAATACC GTCGTGACGT


 


ATTAAGAGAA TGACAGTACG GTAGGCATTC TACGAAAAGA


 


CACTGACCAC TCATGAGTTG GTTCAGTAAG ACTCTTATCA


 


GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG


 


GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC


 


ATCATTGGAA AACGTTCTTC CATACGCCGC TGGCTCAACG


 


AGAACGGGCC GCAGTTATGC CCTATTATGG CGCGGTGTAT


 


CGTCTTGAAA TTTTCACGAG TAGTAACCTT TTGCAAGAAG


 


GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC


 


AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG


 


CATCTTTTAC TTTCACCAGC CCCCGCTTTT GAGAGTTCCT


 


AGAATGGCGA CAACTCTAGG TCAAGCTACA TTGGGTGAGC


 


ACGTGGGTTG ACTAGAAGTC GTAGAAAATG AAAGTGGTCG


 


GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA


 


AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT


 


ACTCTTCCTT TTTCAATATT CAAAGACCCA CTCGTTTTTG


 


TCCTTCCGTT TTACGGCGTT TTTTCCCTTA TTCCCGCTGT


 


GCCTTTACAA CTTATGAGTA TGAGAAGGAA AAAGTTATAA


 


ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA


 


CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT


 


CCGCGCACAT TTCCCCGAAA TAACTTCGTA AATAGTCCCA


 


ATAACAGAGT ACTCGCCTAT GTATAAACTT ACATAAATCT


 


TTTTATTTGT TTATCCCCAA GGCGCGTGTA AAGGGGCTTT


 


AGTGCCACCT GACGTCTAAG AAACCATTAT TATCATGACA


 


TTAACCTATA AAAATAGGCG TATCACGAGG CCCTTTCGTC


 


TCACGGTGGA CTGCAGATTC TTTGGTAATA ATAGTACTGT


 


AATTGGATAT TTTTATCCGC ATAGTGCTCC GGGAAAGCAG


 


SEQ ID: 02


GGGGAGCCGC GCCGAAGGCG TGGGGGAACC CCGCAGGGGT


 


GCCCTTCTTT GGGCACCAAA GAACTAGATA TAGGGCGAAA


 


TGCGAAAGAC TTAAAAATCA CCCCTCGGCG CGGCTTCCGC


 


ACCCCCTTGG GGCGTCCCCA CGGGAAGAAA CCCGTGGTTT


 


CTTGATCTAT ATCCCGCTTT ACGCTTTCTG AATTTTTAGT


 


ACAACTTAAA AAAGGGGGGT ACGCAACAGC TCATTGCGGC


 


ACCCCCCGCA ATAGCTCATT GCGTAGGTTA AAGAAAATCT


 


GTAATTGACT GCCACTTTTA TGTTGAATTT TTTCCCCCCA


 


TGCGTTGTCG AGTAACGCCG TGGGGGGCGT TATCGAGTAA


 


CGCATCCAAT TTCTTTTAGA CATTAACTGA CGGTGAAAAT


 


CGCAACGCAT AATTGTTGTC GCGCTGCCGA AAAGTTGCAG


 


CTGATTGCGC ATGGTGCCGC AACCGTGCGG CACCCTACCG


 


CATGGAGATA AGCATGGCCA GCGTTGCGTA TTAACAACAG


 


CGCGACGGCT TTTCAACGTC GACTAACGCG TACCACGGCG


 


TTGGCACGCC GTGGGATGGC GTACCTCTAT TCGTACCGGT


 


CGCAGTCCAG AGAAATCGGC ATTCAAGCCA AGAACAAGCC


 


CGGTCACTGG GTGCAAACGG AACGCAAAGC GCATGAGGCG


 


TGGGCCGGGC TTATTGCGAG GCGTCAGGTC TCTTTAGCCG


 


TAAGTTCGGT TCTTGTTCGG GCCAGTGACC CACGTTTGCC


 


TTGCGTTTCG CGTACTCCGC ACCCGGCCCG AATAACGCTC


 


GAAACCCACG GCGGCAATGC TGCTGCATCA CCTCGTGGCG


 


CAGATGGGCC ACCAGAACGC CGTGGTGGTC AGCCAGAAGA


 


CACTTTCCAA GCTCATCGGA CTTTGGGTGC CGCCGTTACG


 


ACGACGTAGT GGAGCACCGC GTCTACCCGG TGGTCTTGCG


 


GCACCACCAG TCGGTCTTCT GTGAAAGGTT CGAGTAGCCT


 


CGTTCTTTGC GGACGGTCCA ATACGCAGTC AAGGACTTGG


 


TGGCCGAGCG CTGGATCTCC GTCGTGAAGC TCAACGGCCC


 


CGGCACCGTG TCGGCCTACG GCAAGAAACG CCTGCCAGGT


 


TATGCGTCAG TTCCTGAACC ACCGGCTCGC GACCTAGAGG


 


CAGCACTTCG AGTTGCCGGG GCCGTGGCAC AGCCGGATGC


 


TGGTCAATGA CCGCGTGGCG TGGGGCCAGC CCCGCGACCA


 


GTTGCGCCTG TCGGTGTTCA GTGCCGCCGT GGTGGTTGAT


 


CACGACGACC AGGACGAATC ACCAGTTACT GGCGCACCGC


 


ACCCCGGTCG GGGCGCTGGT CAACGCGGAC AGCCACAAGT


 


CACGGCGGCA CCACCAACTA GTGCTGCTGG TCCTGCTTAG


 


GCTGTTGGGG CATGGCGACC TGCGCCGCAT CCCGACCCTG


 


TATCCGGGCG AGCAGCAACT ACCGACCGGC CCCGGCGAGG


 


AGCCGCCCAG CCAGCCCGGC CGACAACCCC GTACCGCTGG


 


ACGCGGCGTA GGGCTGGGAC ATAGGCCCGC TCGTCGTTGA


 


TGGCTGGCCG GGGCCGCTCC TCGGCGGGTC GGTCGGGCCG


 


ATTCCGGGCA TGGAACCAGA CCTGCCAGCC TTGACCGAAA


 


CGGAGGAATG GGAACGGCGC GGGCAGCAGC GCCTGCCGAT


 


GCCCGATGAG CCGTGTTTTC TAAGGCCCGT ACCTTGGTCT


 


GGACGGTCGG AACTGGCTTT GCCTCCTTAC CCTTGCCGCG


 


CCCGTCGTCG CGGACGGCTA CGGGCTACTC GGCACAAAAG


 


TGGACGATGG CGAGCCGTTG GAGCCGCCGA CACGGGTCAC


 


GCTGCCGCGC CGGTAGCACT TGGGTTGCGC AGCAACCCGT


 


AAGTGCGCTG TTCCAGACTA ACCTGCTACC GCTCGGCAAC


 


CTCGGCGGCT GTGCCCAGTG CGACGGCGCG GCCATCGTGA


 


ACCCAACGCG TCGTTGGGCA TTCACGCGAC AAGGTCTGAT


 


TCGGCTGTAG CCGCCTCGCC GCCCTATACC TTGTCTGCCT


 


CCCCGCGTTG CGTCGCGGTG CATGGAGCCG GGCCACCTCG


 


ACCTGAATGG AAGCCGGCGG AGCCGACATC GGCGGAGCGG


 


CGGGATATGG AACAGACGGA GGGGCGCAAC GCAGCGCCAC


 


GTACCTCGGC CCGGTGGAGC TGGACTTACC TTCGGCCGCC


 


CACCTCGCTA ACGGATTCAC CGTTTTTATC AGGCTCTGGG


 


AGGCAGAATA AATGATCATA TCGTCAATTA TTACCTCCAC


 


GGGGAGAGCC TGAGCAAACT GTGGAGCGAT TGCCTAAGTG


 


GCAAAAATAG TCCGAGACCC TCCGTCTTAT TTACTAGTAT


 


AGCAGTTAAT AATGGAGGTG CCCCTCTCGG ACTCGTTTGA


 


GGCCTCAGGC ATTTGAGAAG CACACGGTCA CACTGCTTCC


 


GGTAGTCAAT AAACCGGTAA ACCAGCAATA GACATAAGCG


 


GCTATTTAAC GACCCTGCCC CCGGAGTCCG TAAACTCTTC


 


GTGTGCCAGT GTGACGAAGG CCATCAGTTA TTTGGCCATT


 


TGGTCGTTAT CTGTATTCGC CGATAAATTG CTGGGACGGG


 


TGAACCGACG ACCGGGTCGA ATTTGCTTTC GAATTTCTGC


 


CATTCATCCG CTTATTATCA CTTATTCAGG CGTAGCACCA


 


GGCGTTTAAG GGCACCAATA ACTTGGCTGC TGGCCCAGCT


 


TAAACGAAAG CTTAAAGACG GTAAGTAGGC GAATAATAGT


 


GAATAAGTCC GCATCGTGGT CCGCAAATTC CCGTGGTTAT


 


ACTGCCTTAA AAAAATTACG CCCCGCCCTG CCACTCATCG


 


CAGTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA


 


AATTTAACGC GAATTTTAAC TGACGGAATT TTTTTAATGC


 


GGGGCGGGAC GGTGAGTAGC GTCAGCCGGA TAACCAATTT


 


TTTACTCGAC TAAATTGTTT TTAAATTGCG CTTAAAATTG


 


AAAATATTAA CGCTTACAAT TTCCATTCGC CATTCAGGCT


 


GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG


 


CTATTACGCC AGCTGGCGAA TTTTATAATT GCGAATGTTA


 


AAGGTAAGCG GTAAGTCCGA CGCGTTGACA ACCCTTCCCG


 


CTAGCCACGC CCGGAGAAGC GATAATGCGG TCGACCGCTT


 


AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA


 


GGGTTTTCCC AGTCACGACG TTGTAAAACG ACGGCCAGTG


 


AGCGCGCGTA ATACGACTCA TCCCCCTACA CGACGTTCCG


 


CTAATTCAAC CCATTGCGGT CCCAAAAGGG TCAGTGCTGC


 


AACATTTTGC TGCCGGTCAC TCGCGCGCAT TATGCTGAGT


 


CTATAGGGCG AATTGGAGCT CCACCGCGGT GGCGGCCGCT


 


CTAGAACTAG TGGATCCCCC GGGCTGCAGG AATTCGATAT


 


CAAGCTTATC GATACCGTCG GATATCCCGC TTAACCTCGA


 


GGTGGCGCCA CCGCCGGCGA GATCTTGATC ACCTAGGGGG


 


CCCGACGTCC TTAAGCTATA GTTCGAATAG CTATGGCAGC


 


ACCTCGAGGG GGGGCCCGGT ACCCAGCTTT TGTTCCCTTT


 


AGTGAGGGTT AATTGCGCGC TTGGCGTAAT CATGGTCATA


 


GCTGTTTCCT GTGTGAAATT TGGAGCTCCC CCCCGGGCCA


 


TGGGTCGAAA ACAAGGGAAA TCACTCCCAA TTAACGCGCG


 


AACCGCATTA GTACCAGTAT CGACAAAGGA CACACTTTAA


 


GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG


 


CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA


 


CTCACATTAA TTGCGTTGCG CAATAGGCGA GTGTTAAGGT


 


GTGTTGTATG CTCGGCCTTC GTATTTCACA TTTCGGACCC


 


CACGGATTAC TCACTCGATT GAGTGTAATT AACGCAACGC


 


CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG


 


CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT


 


TGCGTATTGG GCGCATGCAT GAGTGACGGG CGAAAGGTCA


 


GCCCTTTGGA CAGCACGGTC GACGTAATTA CTTAGCCGGT


 


TGCGCGCCCC TCTCCGCCAA ACGCATAACC CGCGTACGTA


 


AAAAACTGTT GTAATTCATT AAGCATTCTG CCGACATGGA


 


AGCCATCACA AACGGCATGA TGAACCTGAA TCGCCAGCGG


 


CATCAGCACC TTGTCGCCTT TTTTTGACAA CATTAAGTAA


 


TTCGTAAGAC GGCTGTACCT TCGGTAGTGT TTGCCGTACT


 


ACTTGGACTT AGCGGTCGCC GTAGTCGTGG AACAGCGGAA


 


GCGTATAATA TTTGCCCATG GGGGTGGGCG AAGAACTCCA


 


GCATGAGATC CCCGCGCTGG AGGATCATCC AGCCGGCGTC


 


CCGGAAAACG ATTCCGAAGC CGCATATTAT AAACGGGTAC


 


CCCCACCCGC TTCTTGAGGT CGTACTCTAG GGGCGCGACC


 


TCCTAGTAGG TCGGCCGCAG GGCCTTTTGC TAAGGCTTCG


 


CCAACCTTTC ATAGAAGGCG GCGGTGGAAT CGAAATCTCG


 


TGATGGCAGG TTGGGCGTCG CTTGGTCGGT CATTTCGAAC


 


CCCAGAGTCC CGCTCAGAAG GGTTGGAAAG TATCTTCCGC


 


CGCCACCTTA GCTTTAGAGC ACTACCGTCC AACCCGCAGC


 


GAACCAGCCA GTAAAGCTTG GGGTCTCAGG GCGAGTCTTC


 


AACTCGTCAA GAAGGCGATA GAAGGCGATG CGCTGCGAAT


 


CGGGAGCGGC GATACCGTAA AGCACGAGGA AGCGGTCAGC


 


CCATTCGCCG CCAAGCTCTT TTGAGCAGTT CTTCCGCTAT


 


CTTCCGCTAC GCGACGCTTA GCCCTCGCCG CTATGGCATT


 


TCGTGCTCCT TCGCCAGTCG GGTAAGCGGC GGTTCGAGAA


 


CAGCAATATC ACGGGTAGCC AACGCTATGT CCTGATAGCG


 


GTCCGCCACA CCCAGCCGGC CACAGTCGAT GAATCCAGAA


 


AAGCGGCCAT TTTCCACCAT GTCGTTATAG TGCCCATCGG


 


TTGCGATACA GGACTATCGC CAGGCGGTGT GGGTCGGCCG


 


GTGTCAGCTA CTTAGGTCTT TTCGCCGGTA AAAGGTGGTA


 


GATATTCGGC AAGCAGGCAT CGCCATGGGT CACGACGAGA


 


TCCTCGCCGT CGGGCATGCG CGCCTTGAGC CTGGCGAACA


 


GTTCGGCTGG CGCGAGCCCC CTATAAGCCG TTCGTCCGTA


 


GCGGTACCCA GTGCTGCTCT AGGAGCGGCA GCCCGTACGC


 


GCGGAACTCG GACCGCTTGT CAAGCCGACC GCGCTCGGGG


 


TGATGCTCTT CGTCCAGATC ATCCTGATCG ACAAGACCGG


 


CTTCCATCCG AGTACGTGCT CGCTCGATGC GATGTTTCGC


 


TTGGTGGTCG AATGGGCAGG ACTACGAGAA GCAGGTCTAG


 


TAGGACTAGC TGTTCTGGCC GAAGGTAGGC TCATGCACGA


 


GCGAGCTACG CTACAAAGCG AACCACCAGC TTACCCGTCC


 


TAGCCGGATC AAGCGTATGC AGCCGCCGCA TTGCATCAGC


 


CATGATGGAT ACTTTCTCGG CAGGAGCAAG GTGAGATGAC


 


AGGAGATCCT GCCCCGGCAC ATCGGCCTAG TTCGCATACG


 


TCGGCGGCGT AACGTAGTCG GTACTACCTA TGAAAGAGCC


 


GTCCTCGTTC CACTCTACTG TCCTCTAGGA CGGGGCCGTG 


 


TTCGCCCAAT AGCAGCCAGT CCCTTCCCGC TTCAGTGACA


 


ACGTCGAGCA CAGCTGCGCA AGGAACGCCC GTCGTGGCCA


 


GCCACGATAG CCGCGCTGCC AAGCGGGTTA TCGTCGGTCA


 


GGGAAGGGCG AAGTCACTGT TGCAGCTCGT GTCGACGCGT


 


TCCTTGCGGG CAGCACCGGT CGGTGCTATC GGCGCGACGG


 


TCGTCCTGCA GTTCATTCAG GGCACCGGAC AGGTCGGTCT


 


TGACAAAAAG AACCGGGCGC CCCTGCGCTG ACAGCCGGAA


 


CACGGCGGCA TCAGAGCAGC AGCAGGACGT CAAGTAAGTC


 


CCGTGGCCTG TCCAGCCAGA ACTGTTTTTC TTGGCCCGCG


 


GGGACGCGAC TGTCGGCCTT GTGCCGCCGT AGTCTCGTCG


 


CGATTGTCTG TTGTGCCCAG TCATAGCCGA ATAGCCTCTC


 


CACCCAAGCG GCCGGAGAAC CTGCGTGCAA TCCATCTTGT


 


TCAATCATGC GAAACGATCC GCTAACAGAC AACACGGGTC


 


AGTATCGGCT TATCGGAGAG GTGGGTTCGC CGGCCTCTTG


 


GACGCACGTT AGGTAGAACA AGTTAGTACG CTTTGCTAGG


 


TCATCCTGTC TCTTGATCAG ATCTTGATCC CCTGCGCCAT


 


CAGATCCTTG GCGGCAAGAA AGCCATCCAG TTTACTTTGC


 


AGGGCTTCCC AACCTTACCA AGTAGGACAG AGAACTAGTC


 


TAGAACTAGG GGACGCGGTA GTCTAGGAAC CGCCGTTCTT


 


TCGGTAGGTC AAATGAAACG TCCCGAAGGG TTGGAATGGT


 


GAGGGCGCCC CAGCTGGCAA TTCCGGTTCG CTTGCTGTCC


 


ATAAAACCGC CCAGTCTAGC TATCGCCATG TAAGCCCACT


 


GCAAGCTACC TGCTTTCTCT CTCCCGCGGG GTCGACCGTT


 


AAGGCCAAGC GAACGACAGG TATTTTGGCG GGTCAGATCG


 


ATAGCGGTAC ATTCGGGTGA CGTTCGATGG ACGAAAGAGA


 


TTGCGCTTGC GTTTTCCCTT GTCCAGATAG CCCAGTAGCT


 


GACATTCATC CCAGGTGGCA CTTTTCGGGG AAATGTGCGC


 


GCCCGCGTTC CTGCTGGCGC AACGCGAACG CAAAAGGGAA


 


CAGGTCTATC GGGTCATCGA CTGTAAGTAG GGTCCACCGT


 


GAAAAGCCCC TTTACACGCG CGGGCGCAAG GACGACCGCG


 


TGGGCCTGTT TCTGGCGCTG GACTTCCCGC TGTTCCGTCA


 


GCAGCTTTTC GCCCACGGCC TTGATGATCG CGGCGGCCTT


 


GGCCTGCATA TCCCGATTCA ACCCGGACAA AGACCGCGAC


 


CTGAAGGGCG ACAAGGCAGT CGTCGAAAAG CGGGTGCCGG


 


AACTACTAGC GCCGCCGGAA CCGGACGTAT AGGGCTAAGT


 


ACGGCCCCAG GGCGTCCAGA ACGGGCTTCA GGCGCTCCCG


 


AAGGTCTCGG GCCGTCTCTT GGGCTTGATC GGCCTTCTTG


 


CGCATCTCAC GCGCTCCTGC TGCCGGGGTC CCGCAGGTCT


 


TGCCCGAAGT CCGCGAGGGC TTCCAGAGCC CGGCAGAGAA


 


CCCGAACTAG CCGGAAGAAC GCGTAGAGTG CGCGAGGACG


 


GGCGGCCTGT AGGGCAGGCT CATACCCCTG CCGAACCGCT


 


TTTGTCAGCC GGTCGGCCAC GGCTTCCGGC GTCTCAACGC


 


GCTTTGAGAT TCCCAGCTTT CCGCCGGACA TCCCGTCCGA


 


GTATGGGGAC GGCTTGGCGA AAACAGTCGG CCAGCCGGTG


 


CCGAAGGCCG CAGAGTTGCG CGAAACTCTA AGGGTCGAAA


 


TCGGCCAATC CCTGCGGTGC ATAGGCGCGT GGCTCGACCG


 


CTTGCGGGCT GATGGTGACG TGGCCCACTG GTGGCCGCTC


 


CAGGGCCTCG TAGAACGCCT AGCCGGTTAG GGACGCCACG


 


TATCCGCGCA CCGAGCTGGC GAACGCCCGA CTACCACTGC


 


ACCGGGTGAC CACCGGCGAG GTCCCGGAGC ATCTTGCGGA


 


GAATGCGCGT GTGACGTGCC TTGCTGCCCT CGATGCCCCG


 


TTGCAGCCCT AGATCGGCCA CAGCGGCCGC AAACGTGGTC


 


TGGTCGCGGG TCATCTGCGC CTTACGCGCA CACTGCACGG


 


AACGACGGGA GCTACGGGGC AACGTCGGGA TCTAGCCGGT


 


GTCGCCGGCG TTTGCACCAG ACCAGCGCCC AGTAGACGCG


 


TTTGTTGCCG ATGAACTCCT TGGCCGACAG CCTGCCGTCC


 


TGCGTCAGCG GCACCACGAA CGCGGTCATG TGCGGGCTGG


 


TTTCGTCACG GTGGATGCTG AAACAACGGC TACTTGAGGA


 


ACCGGCTGTC GGACGGCAGG ACGCAGTCGC CGTGGTGCTT


 


GCGCCAGTAC ACGCCCGACC AAAGCAGTGC CACCTACGAC


 


GCCGTCACGA TGCGATCCGC CCCGTACTTG TCCGCCAGCC


 


ACTTGTGCGC CTTCTCGAAG AACGCCGCCT GCTGTTCTTG


 


GCTGGCCGAC TTCCACCATT CGGCAGTGCT ACGCTAGGCG


 


GGGCATGAAC AGGCGGTCGG TGAACACGCG GAAGAGCTTC


 


TTGCGGCGGA CGACAAGAAC CGACCGGCTG AAGGTGGTAA


 


CCGGGCTGGC CGTCATGACG TACTCGACCG CCAACACAGC


 


GTCCTTGCGC CGCTTCTCTG GCAGCAACTC GCGCAGTCGG


 


CCCATCGCTT CATCGGTGCT GGCCCGACCG GCAGTACTGC


 


ATGAGCTGGC GGTTGTGTCG CAGGAACGCG GCGAAGAGAC


 


CGTCGTTGAG CGCGTCAGCC GGGTAGCGAA GTAGCCACGA


 


GCTGGCCGCC CAGTGCTCGT TCTCTGGCGT CCTGCTGGCG


 


TCAGCGTTGG GCGTCTCGCG CTCGCGGTAG GCGTGCTTGA


 


GACTGGCCGC CACGTTGCCC CGACCGGCGG GTCACGAGCA


 


AGAGACCGCA GGACGACCGC AGTCGCAACC CGCAGAGCGC


 


GAGCGCCATC CGCACGAACT CTGACCGGCG GTGCAACGGG


 


ATTTTCGCCA GCTTCTTGCA TCGCATGATC GCGTATGCCG


 


CCATGCCTGC CCCTCCCTTT TGGTGTCCAA CCGGCTCGAC


 


GGGGGCAGCG CAAGGCGGTG TAAAAGCGGT CGAAGAACGT


 


AGCGTACTAG CGCATACGGC GGTACGGACG GGGAGGGAAA


 


ACCACAGGTT GGCCGAGCTG CCCCCGTCGC GTTCCGCCAC


 


CCTCCGGCGG GCCACTCAAT GCTTGAGTAT ACTCACTAGA


 


CTTTGCTTCG CAAAGTCGTG ACCGCCTACG GCGGCTGCGG


 


CGCCCTACGG GCTTGCTCTC GGAGGCCGCC CGGTGAGTTA


 


CGAACTCATA TGAGTGATCT GAAACGAAGC GTTTCAGCAC


 


TGGCGGATGC CGCCGACGCC GCGGGATGCC CGAACGAGAG


 


CGGGCTTCGC CCTGCGCGGT CGCTGCGCTC CCTTGCCAGC


 


CCGTGGATAT GTGGACGATG GCCGCGAGCG GCCACCGGCT


 


GGCTCGCTTC GCTCGGCCCG GCCCGAAGCG GGACGCGCCA


 


GCGACGCGAG GGAACGGTCG GGCACCTATA CACCTGCTAC


 


CGGCGCTCGC CGGTGGCCGA CCGAGCGAAG CGAGCCGGGC


 


TGGACAACCC TGCTGGACAA GCTGATGGAC AGGCTGCGCC


 


TGCCCACGAG CTTGACCACA GGGATTGCCC ACCGGCTACC


 


CAGCCTTCGA CCACATACCC ACCTGTTGGG ACGACCTGTT


 


CGACTACCTG TCCGACGCGG ACGGGTGCTC GAACTGGTGT


 


CCCTAACGGG TGGCCGATGG GTCGGAAGCT GGTGTATGGG


 


ACCGGCTCCA ACTGCGCGGC CTGCGGCCTT GCCCCATCAA


 


TTTTTTTAAT TTTCTCTGGG GAAAAGCCTC CGGCCTGCGG


 


CCTGCGCGCT TCGCTTGCCG TGGCCGAGGT TGACGCGCCG


 


GACGCCGGAA CGGGGTAGTT AAAAAAATTA AAAGAGACCC


 


CTTTTCGGAG GCCGGACGCC GGACGCGCGA AGCGAACGGC


 


GTTGGACACC AAGTGGAAGG CGGGTCAAGG CTCGCGCAGC


 


GACCGCGCAG CGGCTTGGCC TTGACGCGCC TGGAACGACC


 


CAAGCCTATG CGAGTGGGGG CAACCTGTGG TTCACCTTCC


 


GCCCAGTTCC GAGCGCGTCG CTGGCGCGTC GCCGAACCGG


 


AACTGCGCGG ACCTTGCTGG GTTCGGATAC GCTCACCCCC


 


CAGTCGAAGG CGAAGCCCGC CCGCCTGCCC CCCGAGCCTC


 


ACGGCGGCGA GTGCGGGGGT TCCAAGGGGG CAGCGCCACC


 


TTGGGCAAGG CCGAAGGCCG GTCAGCTTCC GCTTCGGGCG


 


GGCGGACGGG GGGCTCGGAG TGCCGCCGCT CACGCCCCCA


 


AGGTTCCCCC GTCGCGGTGG AACCCGTTCC GGCTTCCGGC


 


CGCAGTCGAT CAACAAGCCC CGGAGGGGCC ACTTTTTGCC


 


GGAGGCGTCA GCTAGTTGTT CGGGGCCTCC CCGGTGAAAA


 


ACGGCCTC


 


SEQ ID: 03


GGGGAGCCGC GCCGAAGGCG TGGGGGAACC CCGCAGGGGT


 


GCCCTTCTTT GGGCACCAAA GAACTAGATA TAGGGCGAAA


 


TGCGAAAGAC TTAAAAATCA CCCCTCGGCG CGGCTTCCGC


 


ACCCCCTTGG GGCGTCCCCA CGGGAAGAAA CCCGTGGTTT


 


CTTGATCTAT ATCCCGCTTT ACGCTTTCTG AATTTTTAGT


 


ACAACTTAAA AAAGGGGGGT ACGCAACAGC TCATTGCGGC


 


ACCCCCCGCA ATAGCTCATT GCGTAGGTTA AAGAAAATCT


 


GTAATTGACT GCCACTTTTA TGTTGAATTT TTTCCCCCCA


 


TGCGTTGTCG AGTAACGCCG TGGGGGGCGT TATCGAGTAA


 


CGCATCCAAT TTCTTTTAGA CATTAACTGA CGGTGAAAAT


 


CGCAACGCAT AATTGTTGTC GCGCTGCCGA AAAGTTGCAG


 


CTGATTGCGC ATGGTGCCGC AACCGTGCGG CACCCTACCG


 


CATGGAGATA AGCATGGCCA GCGTTGCGTA TTAACAACAG


 


CGCGACGGCT TTTCAACGTC GACTAACGCG TACCACGGCG


 


TTGGCACGCC GTGGGATGGC GTACCTCTAT TCGTACCGGT


 


CGCAGTCCAG AGAAATCGGC ATTCAAGCCA AGAACAAGCC


 


CGGTCACTGG GTGCAAACGG AACGCAAAGC GCATGAGGCG


 


TGGGCCGGGC TTATTGCGAG GCGTCAGGTC TCTTTAGCCG


 


TAAGTTCGGT TCTTGTTCGG GCCAGTGACC CACGTTTGCC


 


TTGCGTTTCG CGTACTCCGC ACCCGGCCCG AATAACGCTC


 


GAAACCCACG GCGGCAATGC TGCTGCATCA CCTCGTGGCG


 


CAGATGGGCC ACCAGAACGC CGTGGTGGTC AGCCAGAAGA


 


CACTTTCCAA GCTCATCGGA CTTTGGGTGC CGCCGTTACG


 


ACGACGTAGT GGAGCACCGC GTCTACCCGG TGGTCTTGCG


 


GCACCACCAG TCGGTCTTCT GTGAAAGGTT CGAGTAGCCT


 


CGTTCTTTGC GGACGGTCCA ATACGCAGTC AAGGACTTGG


 


TGGCCGAGCG CTGGATCTCC GTCGTGAAGC TCAACGGCCC


 


CGGCACCGTG TCGGCCTACG GCAAGAAACG CCTGCCAGGT


 


TATGCGTCAG TTCCTGAACC ACCGGCTCGC GACCTAGAGG


 


CAGCACTTCG AGTTGCCGGG GCCGTGGCAC AGCCGGATGC


 


TGGTCAATGA CCGCGTGGCG TGGGGCCAGC CCCGCGACCA


 


GTTGCGCCTG TCGGTGTTCA GTGCCGCCGT GGTGGTTGAT


 


CACGACGACC AGGACGAATC ACCAGTTACT GGCGCACCGC


 


ACCCCGGTCG GGGCGCTGGT CAACGCGGAC AGCCACAAGT


 


CACGGCGGCA CCACCAACTA GTGCTGCTGG TCCTGCTTAG


 


GCTGTTGGGG CATGGCGACC TGCGCCGCAT CCCGACCCTG


 


TATCCGGGCG AGCAGCAACT ACCGACCGGC CCCGGCGAGG


 


AGCCGCCCAG CCAGCCCGGC CGACAACCCC GTACCGCTGG


 


ACGCGGCGTA GGGCTGGGAC ATAGGCCCGC TCGTCGTTGA


 


TGGCTGGCCG GGGCCGCTCC TCGGCGGGTC GGTCGGGCCG


 


ATTCCGGGCA TGGAACCAGA CCTGCCAGCC TTGACCGAAA


 


CGGAGGAATG GGAACGGCGC GGGCAGCAGC GCCTGCCGAT


 


GCCCGATGAG CCGTGTTTTC TAAGGCCCGT ACCTTGGTCT


 


GGACGGTCGG AACTGGCTTT GCCTCCTTAC CCTTGCCGCG


 


CCCGTCGTCG CGGACGGCTA CGGGCTACTC GGCACAAAAG


 


TGGACGATGG CGAGCCGTTG GAGCCGCCGA CACGGGTCAC


 


GCTGCCGCGC CGGTAGCACT TGGGTTGCGC AGCAACCCGT


 


AAGTGCGCTG TTCCAGACTA ACCTGCTACC GCTCGGCAAC


 


CTCGGCGGCT GTGCCCAGTG CGACGGCGCG GCCATCGTGA


 


ACCCAACGCG TCGTTGGGCA TTCACGCGAC AAGGTCTGAT


 


TCGGCTGTAG CCGCCTCGCC GCCCTATACC TTGTCTGCCT


 


CCCCGCGTTG CGTCGCGGTG CATGGAGCCG GGCCACCTCG


 


ACCTGAATGG AAGCCGGCGG AGCCGACATC GGCGGAGCGG


 


CGGGATATGG AACAGACGGA GGGGCGCAAC GCAGCGCCAC


 


GTACCTCGGC CCGGTGGAGC TGGACTTACC TTCGGCCGCC


 


CACCTCGCTA ACGGATTCAC CGTTTTTATC AGGCTCTGGG


 


AGGCAGAATA AATGATCATA TCGTCAATTA TTACCTCCAC


 


GGGGAGAGCC TGAGCAAACT GTGGAGCGAT TGCCTAAGTG


 


GCAAAAATAG TCCGAGACCC TCCGTCTTAT TTACTAGTAT


 


AGCAGTTAAT AATGGAGGTG CCCCTCTCGG ACTCGTTTGA


 


GGCCTCAGGC ATTTGAGAAG CACACGGTCA CACTGCTTCC


 


GGTAGTCAAT AAACCGGTAA ACCAGCAATA GACATAAGCG


 


GCTATTTAAC GACCCTGCCC CCGGAGTCCG TAAACTCTTC


 


GTGTGCCAGT GTGACGAAGG CCATCAGTTA TTTGGCCATT


 


TGGTCGTTAT CTGTATTCGC CGATAAATTG CTGGGACGGG


 


TGAACCGACG ACCGGGTCGA ATTTGCTTTC GAATTTCTGC


 


CATTCATCCG CTTATTATCA CTTATTCAGG CGTAGCACCA


 


GGCGTTTAAG GGCACCAATA ACTTGGCTGC TGGCCCAGCT


 


TAAACGAAAG CTTAAAGACG GTAAGTAGGC GAATAATAGT


 


GAATAAGTCC GCATCGTGGT CCGCAAATTC CCGTGGTTAT


 


ACTGCCTTAA AAAAATTACG CCCCGCCCTG CCACTCATCG


 


CAGTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA


 


AATTTAACGC GAATTTTAAC TGACGGAATT TTTTTAATGC


 


GGGGCGGGAC GGTGAGTAGC GTCAGCCGGA TAACCAATTT


 


TTTACTCGAC TAAATTGTTT TTAAATTGCG CTTAAAATTG


 


AAAATATTAA CGCTTACAAT TTCCATTCGC CATTCAGGCT


 


GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG


 


CTATTACGCC AGCTGGCGAA TTTTATAATT GCGAATGTTA


 


AAGGTAAGCG GTAAGTCCGA CGCGTTGACA ACCCTTCCCG


 


CTAGCCACGC CCGGAGAAGC GATAATGCGG TCGACCGCTT


 


AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA


 


GGGTTTTCCC AGTCACGACG TTGTAAAACG ACGGCCAGTG


 


AGCGCGCGTA ATACGACTCA TCCCCCTACA CGACGTTCCG


 


CTAATTCAAC CCATTGCGGT CCCAAAAGGG TCAGTGCTGC


 


AACATTTTGC TGCCGGTCAC TCGCGCGCAT TATGCTGAGT


 


CTATAGGGCG AATTGGAGCT CCACCGCGGT GGCGGCCGCT


 


CTAGAACTAG TGGATCCCCC GGGCTGCAGG AATTCGATAT


 


CAAGCTTATC GATACCGTCG GATATCCCGC TTAACCTCGA


 


GGTGGCGCCA CCGCCGGCGA GATCTTGATC ACCTAGGGGG


 


CCCGACGTCC TTAAGCTATA GTTCGAATAG CTATGGCAGC


 


ACGGGCCCGG GATCCGATGC TCTTCCGCTA AGATCTTTTA


 


CTAGTTCAGT CCATCTCGCC GTGTATGCGG GCCTGACGGA


 


TCAACGTTCC CACCGAGCCA TGCCCGGGCC CTAGGCTACG


 


AGAAGGCGAT TCTAGAAAAT GATCAAGTCA GGTAGAGCGG


 


CACATACGCC CGGACTGCCT AGTTGCAAGG GTGGCTCGGT


 


GTCGAGATGT TCATCTGGTC GGCGATCTGC CGGTACTTCA


 


AACCTTGTTT GCGCAGTTCC ACAGCCTTCT TGCGGCGTTC


 


CTGCGCACGA GCGATGTAGT CAGCTCTACA AGTAGACCAG


 


CCGCTAGACG GCCATGAAGT TTGGAACAAA CGCGTCAAGG


 


TGTCGGAAGA ACGCCGCAAG GACGCGTGCT CGCTACATCA


 


CGCCTCGGTC TTCGGCGACG AGCCGTTTGA TGGTGCTTTT


 


CGAGACGCCG AACTTGTCAG CCAACTCCTG CGCGGTCTGC


 


GTGCGACGCA TCACGCGTTC GCGGAGCCAG AAGCCGCTGC


 


TCGGCAAACT ACCACGAAAA GCTCTGCGGC TTGAACAGTC


 


GGTTGAGGAC GCGCCAGACG CACGCTGCGT AGTGCGCAAG


 


TGCAGCACCC ATCAGTCCGT CCCCTCTGCT GCTGCGAACA


 


GTGCCGATCG ATCGACCTTC TTGAGCTTCG GCCGCGGCGC


 


GGTGGCGTTC TTCCGTACCG ACGTCGTGGG TAGTCAGGCA


 


GGGGAGACGA CGACGCTTGT CACGGCTAGC TAGCTGGAAG


 


AACTCGAAGC CGGCGCCGCG CCACCGCAAG AAGGCATGGC


 


CTTCCGTTTT TGCGCTGCTG CTCACTTTGC CGCGGCGTGC


 


CTGGATTTTC GAGAACTCGG CGGCGGTGAA GGTGCGGTGG


 


GTCCAGTGGG CGACTGATTT GAAGGCAAAA ACGCGACGAC


 


GAGTGAAACG GCGCCGCACG GACCTAAAAG CTCTTGAGCC


 


GCCGCCACTT CCACGCCACC CAGGTCACCC GCTGACTAAA


 


GCCGATCTGC TCGGCCTCGG CCCGACTCAT GGGGCCGATC


 


CCGTCGTTGG CGTCGAGGGT GAAGTTGGTC AGGGCGGTGA


 


AGTCGGTGAC CATCTGCCGC CGGCTAGACG AGCCGGAGCC


 


GGGCTGAGTA CCCCGGCTAG GGCAGCAACC GCAGCTCCCA


 


CTTCAACCAG TCCCGCCACT TCAGCCACTG GTAGACGGCG


 


CACACAGTGA TCGACGGGTA GTTCTGTTTC CGGATCTCGC


 


GGTAGGCCCA TTCCCGGGTG CGGTCGAACA GTTCGACGTT


 


CCGGCCCGTT TCGGTCCTGA GTGTGTCACT AGCTGCCCAT


 


CAAGACAAAG GCCTAGAGCG CCATCCGGGT AAGGGCCCAC


 


GCCAGCTTGT CAAGCTGCAA GGCCGGGCAA AGCCAGGACT


 


CCTGTGTCTT GCGGCCGTAG TCCGGTGGGG CGGGGAAACG


 


GTCACCGAGC GCTTTTGCGA GGCCTTTGAG CGAGTACGGA


 


TCCGAGGGAC CCCAGACCGT GGACACAGAA CGCCGGCATC


 


AGGCCACCCC GCCCCTTTGC CAGTGGCTCG CGAAAACGCT


 


CCGGAAACTC GCTCATGCCT AGGCTCCCTG GGGTCTGGCA


 


CGTCCAGTGC GGGTGGATCG GGTTCTGGGT GAGCTGCTGC


 


GCGTAGCCCT GATCGGCGCC GACCACCGAG GCGATCAGCC


 


CCTGGTTCAC CCGGTCGTAG GCAGGTCACG CCCACCTAGC


 


CCAAGACCCA CTCGACGACG CGCATCGGGA CTAGCCGCGG


 


CTGGTGGCTC CGCTAGTCGG GGACCAAGTG GGCCAGCATC


 


AGCCGCAGCG GGCCCTGTCG GGCTGCCTGG AGGGTGTAGA


 


CCGGGCTTTC GAGCAGCCAC CACAGGTGCG CGTGCTCGGT


 


CGCGGGATTG ATCGTCATCA TCGGCGTCGC CCGGGACAGC


 


CCGACGGACC TCCCACATCT GGCCCGAAAG CTCGTCGGTG


 


GTGTCCACGC GCACGAGCCA GCGCCCTAAC TAGCAGTAGT


 


CGGTCGGATC GGGCAGATCC GCGTTACGTG CGGCCCACTG


 


CGCCTGGTCG TCGTCCACGT CGAGCACCAA GCCCAACCTG


 


ATCGACGGGG TGCGGGCCGC GCCAGCCTAG CCCGTCTAGG


 


CGCAATGCAC GCCGGGTGAC GCGGACCAGC AGCAGGTGCA


 


GCTCGTGGTT CGGGTTGGAC TAGCTGCCCC ACGCCCGGCG


 


AATGTAGCGG CGGGTGAGCG CCTCCGCGCG CGGCTGCGGC


 


CACTGCCCGT CCCGGACGTA GTCATCCGTC GCGTGCGGGT


 


ATTTGAACCG CCAGCGGTCC TTACATCGCC GCCCACTCGC


 


GGAGGCGCGC GCCGACGCCG GTGACGGGCA GGGCCTGCAT


 


CAGTAGGCAG CGCACGCCCA TAAACTTGGC GGTCGCCAGG


 


AACCAGGCGT CAACAGCAGC GGTCATGACC GCCAAGCTAG


 


GGCCGGATCT GTACCGATCG GGGGAGGCGC GCCGCAAATT


 


ATTTAAGAGT CTCGCTAGCA TTGGTCCGCA GTTGTCGTCG


 


CCAGTACTGG CGGTTCGATC CCGGCCTAGA CATGGCTAGC


 


CCCCTCCGCG CGGCGTTTAA TAAATTCTCA GAGCGATCGT


 


AACCATGTCA GGTGTTGCGG TGGGTTCCGG GTAAACCTCC


 


ACCCGAATTA TTTAAGAGTC TCGCTAGCTA AGCCCTATCT


 


GATGCTGCGC GGGGGGTCCT TTGGTACAGT CCACAACGCC


 


ACCCAAGGCC CATTTGGAGG TGGGCTTAAT AAATTCTCAG


 


AGCGATCGAT TCGGGATAGA CTACGACGCG CCCCCCAGGA


 


TCGCACTGAA TCTCAAAGGT GGCCGGCTGA ATTTCGTCGC


 


GCGAAAACCT CCCTGGACAG TTCTGGAATT CAGCAAGAGG


 


TGTGTCTGAA CTTCGGTGTT AGCGTGACTT AGAGTTTCCA


 


CCGGCCGACT TAAAGCAGCG CGCTTTTGGA GGGACCTGTC


 


AAGACCTTAA GTCGTTCTCC ACACAGACTT GAAGCCACAA


 


TTTTTGGGGG GTGACTCCAG CGGGGTGGGC ACAACGCGAA


 


CAGAGACCTT GTGTGTACGA CGGCGGGAGG TAAGTCGGGT


 


ACGGCTCGGA CTGCGGTAGA AAAAACCCCC CACTGAGGTC


 


GCCCCACCCG TGTTGCGCTT GTCTCTGGAA CACACATGCT


 


GCCGCCCTCC ATTCAGCCCA TGCCGAGCCT GACGCCATCT


 


GCAACCGTCG AATCGATTTC GAGCAGAGCG AGCAGAGCAA


 


GATATTCCAA AACTCCGGGG TTCCTCGGCG GCCTCCCCCG


 


TCTGTTTGCT CAACCGAGGG CGTTGGCAGC TTAGCTAAAG


 


CTCGTCTCGC TCGTCTCGTT CTATAAGGTT TTGAGGCCCC


 


AAGGAGCCGC CGGAGGGGGC AGACAAACGA GTTGGCTCCC


 


AGACCTGGCG GTCCCGCGTT TCCGGACGCG CGGGACCGCC


 


TACCGCTCGA GAGCGGAAGA GCATCTAGAT GCATTCGCGA


 


GGTACCCAGC TTTTGTTCCC TCTGGACCGC CAGGGCGCAA


 


AGGCCTGCGC GCCCTGGCGG ATGGCGAGCT CTCGCCTTCT


 


CGTAGATCTA CGTAAGCGCT CCATGGGTCG AAAACAAGGG


 


TTTAGTGAGG GTTAATTGCG CGCTTGGCGT AATCATGGTC


 


ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT


 


CCACACAACA TACGAGCCGG AAATCACTCC CAATTAACGC


 


GCGAACCGCA TTAGTACCAG TATCGACAAA GGACACACTT


 


TAACAATAGG CGAGTGTTAA GGTGTGTTGT ATGCTCGGCC


 


AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC


 


TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC


 


AGTCGGGAAA CCTGTCGTGC TTCGTATTTC ACATTTCGGA


 


CCCCACGGAT TACTCACTCG ATTGAGTGTA ATTAACGCAA


 


CGCGAGTGAC GGGCGAAAGG TCAGCCCTTT GGACAGCACG


 


CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG


 


GTTTGCGTAT TGGGCGCATG CATAAAAACT GTTGTAATTC


 


ATTAAGCATT CTGCCGACAT GTCGACGTAA TTACTTAGCC


 


GGTTGCGCGC CCCTCTCCGC CAAACGCATA ACCCGCGTAC


 


GGAAGCCATC ACAAACGGCA TGATGAACCT GAATCGCCAG


 


CGGCATCAGC ACCTTGTCGC CTTGCGTATA ATATTTGCCC


 


ATGGGGGTGG GCGAAGAACT CCTTCGGTAG TGTTTGCCGT


 


ACTACTTGGA CTTAGCGGTC GCCGTAGTCG TGGAACAGCG


 


GAACGCATAT TATAAACGGG TACCCCCACC CGCTTCTTGA


 


CCAGCATGAG ATCCCCGCGC TGGAGGATCA TCCAGCCGGC


 


GTCCCGGAAA ACGATTCCGA AGCCCAACCT TTCATAGAAG


 


GCGGCGGTGG AATCGAAATC GGTCGTACTC TAGGGGCGCG


 


ACCTCCTAGT AGGTCGGCCG CAGGGCCTTT TGCTAAGGCT


 


TCGGGTTGGA AAGTATCTTC CGCCGCCACC TTAGCTTTAG


 


TCGTGATGGC AGGTTGGGCG TCGCTTGGTC GGTCATTTCG


 


AACCCCAGAG TCCCGCTCAG AAGAACTCGT CAAGAAGGCG


 


ATAGAAGGCG ATGCGCTGCG AGCACTACCG TCCAACCCGC


 


AGCGAACCAG CCAGTAAAGC TTGGGGTCTC AGGGCGAGTC


 


TTCTTGAGCA GTTCTTCCGC TATCTTCCGC TACGCGACGC


 


AATCGGGAGC GGCGATACCG TAAAGCACGA GGAAGCGGTC


 


AGCCCATTCG CCGCCAAGCT CTTCAGCAAT ATCACGGGTA


 


GCCAACGCTA TGTCCTGATA TTAGCCCTCG CCGCTATGGC


 


ATTTCGTGCT CCTTCGCCAG TCGGGTAAGC GGCGGTTCGA


 


GAAGTCGTTA TAGTGCCCAT CGGTTGCGAT ACAGGACTAT


 


GCGGTCCGCC ACACCCAGCC GGCCACAGTC GATGAATCCA


 


GAAAAGCGGC CATTTTCCAC CATGATATTC GGCAAGCAGG


 


CATCGCCATG GGTCACGACG CGCCAGGCGG TGTGGGTCGG


 


CCGGTGTCAG CTACTTAGGT CTTTTCGCCG GTAAAAGGTG


 


GTACTATAAG CCGTTCGTCC GTAGCGGTAC CCAGTGCTGC


 


AGATCCTCGC CGTCGGGCAT GCGCGCCTTG AGCCTGGCGA


 


ACAGTTCGGC TGGCGCGAGC CCCTGATGCT CTTCGTCCAG


 


ATCATCCTGA TCGACAAGAC TCTAGGAGCG GCAGCCCGTA


 


CGCGCGGAAC TCGGACCGCT TGTCAAGCCG ACCGCGCTCG


 


GGGACTACGA GAAGCAGGTC TAGTAGGACT AGCTGTTCTG


 


CGGCTTCCAT CCGAGTACGT GCTCGCTCGA TGCGATGTTT


 


CGCTTGGTGG TCGAATGGGC AGGTAGCCGG ATCAAGCGTA


 


TGCAGCCGCC GCATTGCATC GCCGAAGGTA GGCTCATGCA


 


CGAGCGAGCT ACGCTACAAA GCGAACCACC AGCTTACCCG


 


TCCATCGGCC TAGTTCGCAT ACGTCGGCGG CGTAACGTAG


 


AGCCATGATG GATACTTTCT CGGCAGGAGC AAGGTGAGAT


 


GACAGGAGAT CCTGCCCCGG CACTTCGCCC AATAGCAGCC


 


AGTCCCTTCC CGCTTCAGT TCGGTACTAC CTATGAAAGA


 


GCCGTCCTCG TTCCACTCTA CTGTCCTCTA GGACGGGGCC


 


GTGAAGCGGG TTATCGTCGG TCAGGGAAGG GCGAAGTCAC


 


ACAACGTCGA GCACAGCTGC GCAAGGAACG CCCGTCGTGG


 


CCAGCCACGA TAGCCGCGCT GCCTCGTCCT GCAGTTCATT


 


CAGGGCACCG GACAGGTCGG TGTTGCAGCT CGTGTCGACG


 


CGTTCCTTGC GGGCAGCACC GGTCGGTGCT ATCGGCGCGA


 


CGGAGCAGGA CGTCAAGTAA GTCCCGTGGC CTGTCCAGCC


 


TCTTGACAAA AAGAACCGGG CGCCCCTGCG CTGACAGCCG


 


GAACACGGCG GCATCAGAGC AGCCGATTGT CTGTTGTGCC


 


CAGTCATAGC CGAATAGCCT AGAACTGTTT TTCTTGGCCC


 


GCGGGGACGC GACTGTCGGC CTTGTGCCGC CGTAGTCTCG


 


TCGGCTAACA GACAACACGG GTCAGTATCG GCTTATCGGA


 


CTCCACCCAA GCGGCCGGAG AACCTGCGTG CAATCCATCT


 


TGTTCAATCA TGCGAAACGA TCCTCATCCT GTCTCTTGAT


 


CAGATCTTGA TCCCCTGCGC GAGGTGGGTT CGCCGGCCTC


 


TTGGACGCAC GTTAGGTAGA ACAAGTTAGT ACGCTTTGCT


 


AGGAGTAGGA CAGAGAACTA GTCTAGAACT AGGGGACGCG


 


CATCAGATCC TTGGCGGCAA GAAAGCCATC CAGTTTACTT


 


TGCAGGGCTT CCCAACCTTA CCAGAGGGCG CCCCAGCTGG


 


CAATTCCGGT TCGCTTGCTG GTAGTCTAGG AACCGCCGTT


 


CTTTCGGTAG GTCAAATGAA ACGTCCCGAA GGGTTGGAAT


 


GGTCTCCCGC GGGGTCGACC GTTAAGGCCA AGCGAACGAC


 


TCCATAAAAC CGCCCAGTCT AGCTATCGCC ATGTAAGCCC


 


ACTGCAAGCT ACCTGCTTTC TCTTTGCGCT TGCGTTTTCC


 


CTTGTCCAGA TAGCCCAGTA AGGTATTTTG GCGGGTCAGA


 


TCGATAGCGG TACATTCGGG TGACGTTCGA TGGACGAAAG


 


AGAAACGCGA ACGCAAAAGG GAACAGGTCT ATCGGGTCAT


 


GCTGACATTC ATCCCAGGTG GCACTTTTCG GGGAAATGTG


 


CGCGCCCGCG TTCCTGCTGG CGCTGGGCCT GTTTCTGGCG


 


CTGGACTTCC CGCTGTTCCG CGACTGTAAG TAGGGTCCAC


 


CGTGAAAAGC CCCTTTACAC GCGCGGGCGC AAGGACGACC


 


GCGACCCGGA CAAAGACCGC GACCTGAAGG GCGACAAGGC


 


TCAGCAGCTT TTCGCCCACG GCCTTGATGA TCGCGGCGGC


 


CTTGGCCTGC ATATCCCGAT TCAACGGCCC CAGGGCGTCC


 


AGAACGGGCT TCAGGCGCTC AGTCGTCGAA AAGCGGGTGC


 


CGGAACTACT AGCGCCGCCG GAACCGGACG TATAGGGCTA


 


AGTTGCCGGG GTCCCGCAGG TCTTGCCCGA AGTCCGCGA


 


CCGAAGGTCT CGGGCCGTCT CTTGGGCTTG ATCGGCCTTC


 


TTGCGCATCT CACGCGCTCC TGCGGCGGCC TGTAGGGCAG


 


GCTCATACCC CTGCCGAACC GGCTTCCAGA GCCCGGCAGA


 


GAACCCGAAC TAGCCGGAAG AACGCGTAGA GTGCGCGAGG


 


ACGCCGCCGG ACATCCCGTC CGAGTATGGG GACGGCTTGG


 


GCTTTTGTCA GCCGGTCGGC CACGGCTTCC GGCGTCTCAA


 


CGCGCTTTGA GATTCCCAGC TTTTCGGCCA ATCCCTGCGG


 


TGCATAGGCG CGTGGCTCGA CGAAAACAGT CGGCCAGCCG


 


GTGCCGAAGG CCGCAGAGTT GCGCGAAACT CTAAGGGTCG


 


AAAAGCCGGT TAGGGACGCC ACGTATCCGC GCACCGAGCT


 


CCGCTTGCGG GCTGATGGTG ACGTGGCCCA CTGGTGGCCG


 


CTCCAGGGCC TCGTAGAACG CCTGAATGCG CGTGTGACGT


 


GCCTTGCTGC CCTCGATGCC GGCGAACGCC CGACTACCAC


 


TGCACCGGGT GACCACCGGC GAGGTCCCGG AGCATCTTGC


 


GGACTTACGC GCACACTGCA CGGAACGACG GGAGCTACGG


 


CCGTTGCAGC CCTAGATCGG CCACAGCGGC CGCAAACGTG


 


GTCTGGTCGC GGGTCATCTG CGCTTTGTTG CCGATGAACT


 


CCTTGGCCGA CAGCCTGCCG GGCAACGTCG GGATCTAGCC


 


GGTGTCGCCG GCGTTTGCAC CAGACCAGCG CCCAGTAGAC


 


GCGAAACAAC GGCTACTTGA GGAACCGGCT GTCGGACGGC


 


TCCTGCGTCA GCGGCACCAC GAACGCGGTC ATGTGCGGGC


 


TGGTTTCGTC ACGGTGGATG CTGGCCGTCA CGATGCGATC


 


CGCCCCGTAC TTGTCCGCCA AGGACGCAGT CGCCGTGGTG


 


CTTGCGCCAG TACACGCCCG ACCAAAGCAG TGCCACCTAC


 


GACCGGCAGT GCTACGCTAG GCGGGGCATG AACAGGCGGT


 


GCCACTTGTG CGCCTTCTCG AAGAACGCCG CCTGCTGTTC


 


TTGGCTGGCC GACTTCCACC ATTCCGGGCT GGCCGTCATG


 


ACGTACTCGA CCGCCAACAC CGGTGAACAC GCGGAAGAGC


 


TTCTTGCGGC GGACGACAAG AACCGACCGG CTGAAGGTGG


 


TAAGGCCCGA CCGGCAGTAC TGCATGAGCT GGCGGTTGTG


 


AGCGTCCTTG CGCCGCTTCT CTGGCAGCAA CTCGCGCAGT


 


CGGCCCATCG CTTCATCGGT GCTGCTGGCC GCCCAGTGCT


 


CGTTCTCTGG CGTCCTGCTG TCGCAGGAAC GCGGCGAAGA


 


GACCGTCGTT GAGCGCGTCA GCCGGGTAGC GAAGTAGCCA


 


CGACGACCGG CGGGTCACGA GCAAGAGACC GCAGGACGAC


 


GCGTCAGCGT TGGGCGTCTC GCGCTCGCGG TAGGCGTGCT


 


TGAGACTGGC CGCCACGTTG CCCATTTTCG CCAGCTTCTT


 


GCATCGCATG ATCGCGTATG CGCAGTCGCA ACCCGCAGAG


 


CGCGAGCGCC ATCCGCACGA ACTCTGACCG GCGGTGCAAC


 


GGGTAAAAGC GGTCGAAGAA CGTAGCGTAC TAGCGCATAC


 


CCGCCATGCC TGCCCCTCCC TTTTGGTGTC CAACCGGCTC


 


GACGGGGGCA GCGCAAGGCG GTGCCTCCGG CGGGCCACTC


 


AATGCTTGAG TATACTCACT GGCGGTACGG ACGGGGAGGG


 


AAAACCACAG GTTGGCCGAG CTGCCCCCGT CGCGTTCCGC


 


CACGGAGGCC GCCCGGTGAG TTACGAACTC ATATGAGTGA


 


AGACTTTGCT TCGCAAAGTC GTGACCGCCT ACGGCGGCTG


 


CGGCGCCCTA CGGGCTTGCT CTCCGGGCTT CGCCCTGCGC


 


GGTCGCTGCG CTCCCTTGCC TCTGAAACGA AGCGTTTCAG


 


CACTGGCGGA TGCCGCCGAC GCCGCGGGAT GCCCGAACGA


 


GAGGCCCGAA GCGGGACGCG CCAGCGACGC GAGGGAACGG


 


SEQ ID: 04


GGGGAGCCGC GCCGAAGGCG TGGGGGAACC CCGCAGGGGT


 


GCCCTTCTTT GGGCACCAAA GAACTAGATA TAGGGCGAAA


 


TGCGAAAGAC TTAAAAATCA CCCCTCGGCG CGGCTTCCGC


 


ACCCCCTTGG GGCGTCCCCA CGGGAAGAAA CCCGTGGTTT


 


CTTGATCTAT ATCCCGCTTT ACGCTTTCTG AATTTTTAGT


 


ACAACTTAAA AAAGGGGGGT ACGCAACAGC TCATTGCGGC


 


ACCCCCCGCA ATAGCTCATT GCGTAGGTTA AAGAAAATCT


 


GTAATTGACT GCCACTTTTA TGTTGAATTT TTTCCCCCCA


 


TGCGTTGTCG AGTAACGCCG TGGGGGGCGT TATCGAGTAA


 


CGCATCCAAT TTCTTTTAGA CATTAACTGA CGGTGAAAAT


 


CGCAACGCAT AATTGTTGTC GCGCTGCCGA AAAGTTGCAG


 


CTGATTGCGC ATGGTGCCGC AACCGTGCGG CACCCTACCG


 


CATGGAGATA AGCATGGCCA GCGTTGCGTA TTAACAACAG


 


CGCGACGGCT TTTCAACGTC GACTAACGCG TACCACGGCG


 


TTGGCACGCC GTGGGATGGC GTACCTCTAT TCGTACCGGT


 


CGCAGTCCAG AGAAATCGGC ATTCAAGCCA AGAACAAGCC


 


CGGTCACTGG GTGCAAACGG AACGCAAAGC GCATGAGGCG


 


TGGGCCGGGC TTATTGCGAG GCGTCAGGTC TCTTTAGCCG


 


TAAGTTCGGT TCTTGTTCGG GCCAGTGACC CACGTTTGCC


 


TTGCGTTTCG CGTACTCCGC ACCCGGCCCG AATAACGCTC


 


GAAACCCACG GCGGCAATGC TGCTGCATCA CCTCGTGGCG


 


CAGATGGGCC ACCAGAACGC CGTGGTGGTC AGCCAGAAGA


 


CACTTTCCAA GCTCATCGGA CTTTGGGTGC CGCCGTTACG


 


ACGACGTAGT GGAGCACCGC GTCTACCCGG TGGTCTTGCG


 


GCACCACCAG TCGGTCTTCT GTGAAAGGTT CGAGTAGCCT


 


CGTTCTTTGC GGACGGTCCA ATACGCAGTC AAGGACTTGG


 


TGGCCGAGCG CTGGATCTCC GTCGTGAAGC TCAACGGCCC


 


CGGCACCGTG TCGGCCTACG GCAAGAAACG CCTGCCAGGT


 


TATGCGTCAG TTCCTGAACC ACCGGCTCGC GACCTAGAGG


 


CAGCACTTCG AGTTGCCGGG GCCGTGGCAC AGCCGGATGC


 


TGGTCAATGA CCGCGTGGCG TGGGGCCAGC CCCGCGACCA


 


GTTGCGCCTG TCGGTGTTCA GTGCCGCCGT GGTGGTTGAT


 


CACGACGACC AGGACGAATC ACCAGTTACT GGCGCACCGC


 


ACCCCGGTCG GGGCGCTGGT CAACGCGGAC AGCCACAAGT


 


CACGGCGGCA CCACCAACTA GTGCTGCTGG TCCTGCTTAG


 


GCTGTTGGGG CATGGCGACC TGCGCCGCAT CCCGACCCTG


 


TATCCGGGCG AGCAGCAACT ACCGACCGGC CCCGGCGAGG


 


AGCCGCCCAG CCAGCCCGGC CGACAACCCC GTACCGCTGG


 


ACGCGGCGTA GGGCTGGGAC ATAGGCCCGC TCGTCGTTGA


 


TGGCTGGCCG GGGCCGCTCC TCGGCGGGTC GGTCGGGCCG


 


ATTCCGGGCA TGGAACCAGA CCTGCCAGCC TTGACCGAAA


 


CGGAGGAATG GGAACGGCGC GGGCAGCAGC GCCTGCCGAT


 


GCCCGATGAG CCGTGTTTTC TAAGGCCCGT ACCTTGGTCT


 


GGACGGTCGG AACTGGCTTT GCCTCCTTAC CCTTGCCGCG


 


CCCGTCGTCG CGGACGGCTA CGGGCTACTC GGCACAAAAG


 


TGGACGATGG CGAGCCGTTG GAGCCGCCGA CACGGGTCAC


 


GCTGCCGCGC CGGTAGCACT TGGGTTGCGC AGCAACCCGT


 


AAGTGCGCTG TTCCAGACTA ACCTGCTACC GCTCGGCAAC


 


CTCGGCGGCT GTGCCCAGTG CGACGGCGCG GCCATCGTGA


 


ACCCAACGCG TCGTTGGGCA TTCACGCGAC AAGGTCTGAT


 


TCGGCTGTAG CCGCCTCGCC GCCCTATACC TTGTCTGCCT


 


CCCCGCGTTG CGTCGCGGTG CATGGAGCCG GGCCACCTCG


 


ACCTGAATGG AAGCCGGCGG AGCCGACATC GGCGGAGCGG


 


CGGGATATGG AACAGACGGA GGGGCGCAAC GCAGCGCCAC


 


GTACCTCGGC CCGGTGGAGC TGGACTTACC TTCGGCCGCC


 


CACCTCGCTA ACGGATTCAC CGTTTTTATC AGGCTCTGGG


 


AGGCAGAATA AATGATCATA TCGTCAATTA TTACCTCCAC


 


GGGGAGAGCC TGAGCAAACT GTGGAGCGAT TGCCTAAGTG


 


GCAAAAATAG TCCGAGACCC TCCGTCTTAT TTACTAGTAT


 


AGCAGTTAAT AATGGAGGTG CCCCTCTCGG ACTCGTTTGA


 


GGCCTCAGGC ATTTGAGAAG CACACGGTCA CACTGCTTCC


 


GGTAGTCAAT AAACCGGTAA ACCAGCAATA GACATAAGCG


 


GCTATTTAAC GACCCTGCCC CCGGAGTCCG TAAACTCTTC


 


GTGTGCCAGT GTGACGAAGG CCATCAGTTA TTTGGCCATT


 


TGGTCGTTAT CTGTATTCGC CGATAAATTG CTGGGACGGG


 


TGAACCGACG ACCGGGTCGA ATTTGCTTTC GAATTTCTGC


 


CATTCATCCG CTTATTATCA CTTATTCAGG CGTAGCACCA


 


GGCGTTTAAG GGCACCAATA ACTTGGCTGC TGGCCCAGCT


 


TAAACGAAAG CTTAAAGACG GTAAGTAGGC GAATAATAGT


 


GAATAAGTCC GCATCGTGGT CCGCAAATTC CCGTGGTTAT


 


ACTGCCTTAA AAAAATTACG CCCCGCCCTG CCACTCATCG


 


CAGTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA


 


AATTTAACGC GAATTTTAAC TGACGGAATT TTTTTAATGC


 


GGGGCGGGAC GGTGAGTAGC GTCAGCCGGA TAACCAATTT


 


TTTACTCGAC TAAATTGTTT TTAAATTGCG CTTAAAATTG


 


AAAATATTAA CGCTTACAAT TTCCATTCGC CATTCAGGCT


 


GCGCAACTGT TGGGAAGGGC GATCGGTGCG GGCCTCTTCG


 


CTATTACGCC AGCTGGCGAA TTTTATAATT GCGAATGTTA


 


AAGGTAAGCG GTAAGTCCGA CGCGTTGACA ACCCTTCCCG


 


CTAGCCACGC CCGGAGAAGC GATAATGCGG TCGACCGCTT


 


AGGGGGATGT GCTGCAAGGC GATTAAGTTG GGTAACGCCA


 


GGGTTTTCCC AGTCACGACG TTGTAAAACG ACGGCCAGTG


 


AGCGCGCGTA ATACGACTCA TCCCCCTACA CGACGTTCCG


 


CTAATTCAAC CCATTGCGGT CCCAAAAGGG TCAGTGCTGC


 


AACATTTTGC TGCCGGTCAC TCGCGCGCAT TATGCTGAGT


 


CTATAGGGCG AATTGGAGCT CCACCGCGGT GGCGGCCGCT


 


CTAGAACTAG TGGATCCCCC GGGCTGCAGG AATTCGATAT


 


CAAGCTTTTA CGCCCCGCCC GATATCCCGC TTAACCTCGA


 


GGTGGCGCCA CCGCCGGCGA GATCTTGATC ACCTAGGGGG


 


CCCGACGTCC TTAAGCTATA GTTCGAAAAT GCGGGGCGGG


 


TGCCACTCAT CGCAGTACTG TTGTAATTCA TTAAGCATTC


 


TGCCGACATG GAAGCCATCA CAAACGGCAT GATGAACCTG


 


AATCGCCAGC GGCATCAGCA ACGGTGAGTA GCGTCATGAC


 


AACATTAAGT AATTCGTAAG ACGGCTGTAC CTTCGGTAGT


 


GTTTGCCGTA CTACTTGGAC TTAGCGGTCG CCGTAGTCGT


 


CCTTGTCGCC TTGCGTATAA TATTTGCCCA TGGTGAAAAC


 


GGGGGCGAAG AAGTTGTCCA TATTGGCCAC GTTTAAATCA


 


AAACTGGTGA AACTCACCCA GGAACAGCGG AACGCATATT


 


ATAAACGGGT ACCACTTTTG CCCCCGCTTC TTCAACAGGT


 


ATAACCGGTG CAAATTTAGT TTTGACCACT TTGAGTGGGT


 


GGGATTGGCT GAGACGAAAA ACATATTCTC AATAAACCCT


 


TTAGGGAAAT AGGCCAGGTT TTCACCGTAA CACGCCACAT


 


CTTGCGAATA TATGTGTAGA CCCTAACCGA CTCTGCTTTT


 


TGTATAAGAG TTATTTGGGA AATCCCTTTA TCCGGTCCAA


 


AAGTGGCATT GTGCGGTGTA GAACGCTTAT ATACACATCT


 


AACTGCCGGA AATCGTCGTG GTATTCACTC CAGAGCGATG


 


AAAACGTTTC AGTTTGCTCA TGGAAAACGG TGTAACAAGG


 


GTGAACACTA TCCCATATCA TTGACGGCCT TTAGCAGCAC


 


CATAAGTGAG GTCTCGCTAC TTTTGCAAAG TCAAACGAGT


 


ACCTTTTGCC ACATTGTTCC CACTTGTGAT AGGGTATAGT


 


CCAGCTCACC GTCTTTCATT GCCATACGAA ATTCCGGATG


 


AGCATTCATC AGGCGGGCAA GAATGTGAAT AAAGGCCGGA


 


TAAAACTTGT GCTTATTTTT GGTCGAGTGG CAGAAAGTAA


 


CGGTATGCTT TAAGGCCTAC TCGTAAGTAG TCCGCCCGTT


 


CTTACACTTA TTTCCGGCCT ATTTTGAACA CGAATAAAAA


 


CTTTACGGTC TTTAAAAAGG CCGTAATATC CAGCTGAACG


 


GTCTGGTTAT AGGTACATTG AGCAACTGAC TGAAATGCCT


 


CAAAATGTTC TTTACGATGC GAAATGCCAG AAATTTTTCC


 


GGCATTATAG GTCGACTTGC CAGACCAATA TCCATGTAAC


 


TCGTTGACTG ACTTTACGGA GTTTTACAAG AAATGCTACG


 


CATTGGGATA TATCAACGGT GGTATATCCA GTGATTTTTT


 


TCTCCATATG GTTAACCTTA ATTAAGGGGT CGACGGGCCC


 


GGGATCCGAT GCTCTTCCGC GTAACCCTAT ATAGTTGCCA


 


CCATATAGGT CACTAAAAAA AGAGGTATAC CAATTGGAAT


 


TAATTCCCCA GCTGCCCGGG CCCTAGGCTA CGAGAAGGCG


 


TAAGATCTTT TACTAGTTCA GTCCATCTCG CCGTGTATGC


 


GGGCCTGACG GATCAACGTT CCCACCGAGC CAGTCGAGAT


 


GTTCATCTGG TCGGCGATCT ATTCTAGAAA ATGATCAAGT


 


CAGGTAGAGC GGCACATACG CCCGGACTGC CTAGTTGCAA


 


GGGTGGCTCG GTCAGCTCTA CAAGTAGACC AGCCGCTAGA


 


GCCGGTACTT CAAACCTTGT TTGCGCAGTT CCACAGCCTT


 


CTTGCGGCGT TCCTGCGCAC GAGCGATGTA GTCGCCTCGG


 


TCTTCGGCGA CGAGCCGTTT CGGCCATGAA GTTTGGAACA


 


AACGCGTCAA GGTGTCGGAA GAACGCCGCA AGGACGCGTG


 


CTCGCTACAT CAGCGGAGCC AGAAGCCGCT GCTCGGCAAA


 


GATGGTGCTT TTCGAGACGC CGAACTTGTC AGCCAACTCC


 


TGCGCGGTCT GCGTGCGACG CATCACGCGT TCTGCAGCAC


 


CCATCAGTCC GTCCCCTCTG CTACCACGAA AAGCTCTGCG


 


GCTTGAACAG TCGGTTGAGG ACGCGCCAGA CGCACGCTGC


 


GTAGTGCGCA AGACGTCGTG GGTAGTCAGG CAGGGGAGAC


 


CTGCTGCGAA CAGTGCCGAT CGATCGACCT TCTTGAGCTT


 


CGGCCGCGGC GCGGTGGCGT TCTTCCGTAC CGCTTCCGTT


 


TTTGCGCTGC TGCTCACTTT GACGACGCTT GTCACGGCTA


 


GCTAGCTGGA AGAACTCGAA GCCGGCGCCG CGCCACCGCA


 


AGAAGGCATG GCGAAGGCAA AAACGCGACG ACGAGTGAAA


 


GCCGCGGCGT GCCTGGATTT TCGAGAACTC GGCGGCGGTG


 


AAGGTGCGGT GGGTCCAGTG GGCGACTGAT TTGCCGATCT


 


GCTCGGCCTC GGCCCGACTC CGGCGCCGCA CGGACCTAAA


 


AGCTCTTGAG CCGCCGCCAC TTCCACGCCA CCCAGGTCAC


 


CCGCTGACTA AACGGCTAGA CGAGCCGGAG CCGGGCTGAG


 


ATGGGGCCGA TCCCGTCGTT GGCGTCGAGG GTGAAGTTGG


 


TCAGGGCGGT GAAGTCGGTG ACCATCTGCC GCCACACAGT


 


GATCGACGGG TAGTTCTGTT TACCCCGGCT AGGGCAGCAA


 


CCGCAGCTCC CACTTCAACC AGTCCCGCCA CTTCAGCCAC


 


TGGTAGACGG CGGTGTGTCA CTAGCTGCCC ATCAAGACAA


 


TCCGGATCTC GCGGTAGGCC CATTCCCGGG TGCGGTCGAA


 


CAGTTCGACG TTCCGGCCCG TTTCGGTCCT GACCTGTGTC


 


TTGCGGCCGT AGTCCGGTGG AGGCCTAGAG CGCCATCCGG


 


GTAAGGGCCC ACGCCAGCTT GTCAAGCTGC AAGGCCGGGC


 


AAAGCCAGGA CTGGACACAG AACGCCGGCA TCAGGCCACC


 


GGCGGGGAAA CGGTCACCGA GCGCTTTTGC GAGGCCTTTG


 


AGCGAGTACG GATCCGAGGG ACCCCAGACC GTCGTCCAGT


 


GCGGGTGGAT CGGGTTCTGG CCGCCCCTTT GCCAGTGGCT


 


CGCGAAAACG CTCCGGAAAC TCGCTCATGC CTAGGCTCCC


 


TGGGGTCTGG CAGCAGGTCA CGCCCACCTA GCCCAAGACC


 


GTGAGCTGCT GCGCGTAGCC CTGATCGGCG CCGACCACCG


 


AGGCGATCAG CCCCTGGTTC ACCCGGTCGT AGAGCCGCAG


 


CGGGCCCTGT CGGGCTGCCT CACTCGACGA CGCGCATCGG


 


GACTAGCCGC GGCTGGTGGC TCCGCTAGTC GGGGACCAAG


 


TGGGCCAGCA TCTCGGCGTC GCCCGGGACA GCCCGACGGA


 


GGAGGGTGTA GACCGGGCTT TCGAGCAGCC ACCACAGGTG


 


CGCGTGCTCG GTCGCGGGAT TGATCGTCAT CACGGTCGGA


 


TCGGGCAGAT CCGCGTTACG CCTCCCACAT CTGGCCCGAA


 


AGCTCGTCGG TGGTGTCCAC GCGCACGAGC CAGCGCCCTA


 


ACTAGCAGTA GTGCCAGCCT AGCCCGTCTA GGCGCAATGC


 


TGCGGCCCAC TGCGCCTGGT CGTCGTCCAC GTCGAGCACC


 


AAGCCCAACC TGATCGACGG GGTGCGGGCC GCAATGTAGC


 


GGCGGGTGAG CGCCTCCGCG ACGCCGGGTG ACGCGGACCA


 


GCAGCAGGTG CAGCTCGTGG TTCGGGTTGG ACTAGCTGCC


 


CCACGCCCGG CGTTACATCG CCGCCCACTC GCGGAGGCGC


 


CGCGGCTGCG GCCACTGCCC GTCCCGGACG TAGTCATCCG


 


TCGCGTGCGG GTATTTGAAC CGCCAGCGGT CCAACCAGGC


 


GTCAACAGCA GCGGTCATGA GCGCCGACGC CGGTGACGGG


 


CAGGGCCTGC ATCAGTAGGC AGCGCACGCC CATAAACTTG


 


GCGGTCGCCA GGTTGGTCCG CAGTTGTCGT CGCCAGTACT


 


CCGCCAAGCT AGGGCCGGAT CTGTACCGAT CGGGGGAGGC


 


GCGCCGCAAA TTATTTAAGA GTCTCGCTAG CAAACCATGT


 


CAGGTGTTGC GGTGGGTTCC GGCGGTTCGA TCCCGGCCTA


 


GACATGGCTA GCCCCCTCCG CGCGGCGTTT AATAAATTCT


 


CAGAGCGATC GTTTGGTACA GTCCACAACG CCACCCAAGG


 


GGGTAAACCT CCACCCGAAT TATTTAAGAG TCTCGCTAGC


 


TAAGCCCTAT CTGATGCTGC GCGGGGGGTC CTTCGCACTG


 


AATCTCAAAG GTGGCCGGCT CCCATTTGGA GGTGGGCTTA


 


ATAAATTCTC AGAGCGATCG ATTCGGGATA GACTACGACG


 


CGCCCCCCAG GAAGCGTGAC TTAGAGTTTC CACCGGCCGA


 


GAATTTCGTC GCGCGAAAAC CTCCCTGGAC AGTTCTGGAA


 


TTCAGCAAGA GGTGTGTCTG AACTTCGGTG TTTTTTTGGG


 


GGGTGACTCC AGCGGGGTGG CTTAAAGCAG CGCGCTTTTG


 


GAGGGACCTG TCAAGACCTT AAGTCGTTCT CCACACAGAC


 


TTGAAGCCAC AAAAAAACCC CCCACTGAGG TCGCCCCACC


 


GCACAACGCG AACAGAGACC TTGTGTGTAC GACGGCGGGA


 


GGTAAGTCGG GTACGGCTCG GACTGCGGTA GAGCAACCGT


 


CGAATCGATT TCGAGCAGAG CGTGTTGCGC TTGTCTCTGG


 


AACACACATG CTGCCGCCCT CCATTCAGCC CATGCCGAGC


 


CTGACGCCAT CTCGTTGGCA GCTTAGCTAA AGCTCGTCTC


 


CGAGCAGAGC AAGATATTCC AAAACTCCGG GGTTCCTCGG


 


CGGCCTCCCC CGTCTGTTTG CTCAACCGAG GGAGACCTGG


 


CGGTCCCGCG TTTCCGGACG GCTCGTCTCG TTCTATAAGG


 


TTTTGAGGCC CCAAGGAGCC GCCGGAGGGG GCAGACAAAC


 


GAGTTGGCTC CCTCTGGACC GCCAGGGCGC AAAGGCCTGC


 


CGCGGGACCG CCTACCGCTC GAGAGCGGAA GAGCATCTAG


 


ATGCATTCGC GAGGTACCCA GCTTTTGTTC CCTTTAGTGA


 


GGGTTAATTG CGCGCTTGGC GCGCCCTGGC GGATGGCGAG


 


CTCTCGCCTT CTCGTAGATC TACGTAAGCG CTCCATGGGT


 


CGAAAACAAG GGAAATCACT CCCAATTAAC GCGCGAACCG


 


GTAATCATGG TCATAGCTGT TTCCTGTGTG AAATTGTTAT


 


CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA


 


AGTGTAAAGC CTGGGGTGCC CATTAGTACC AGTATCGACA


 


AAGGACACAC TTTAACAATA GGCGAGTGTT AAGGTGTGTT


 


GTATGCTCGG CCTTCGTATT TCACATTTCG GACCCCACGG


 


TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC


 


TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA


 


TTAATGAATC GGCCAACGCG ATTACTCACT CGATTGAGTG


 


TAATTAACGC AACGCGAGTG ACGGGCGAAA GGTCAGCCCT


 


TTGGACAGCA CGGTCGACGT AATTACTTAG CCGGTTGCGC


 


CGGGGAGAGG CGGTTTGCGT ATTGGGCGCA TGCATAAAAA


 


CTGTTGTAAT TCATTAAGCA TTCTGCCGAC ATGGAAGCCA


 


TCACAAACGG CATGATGAAC GCCCCTCTCC GCCAAACGCA


 


TAACCCGCGT ACGTATTTTT GACAACATTA AGTAATTCGT


 


AAGACGGCTG TACCTTCGGT AGTGTTTGCC GTACTACTTG


 


CTGAATCGCC AGCGGCATCA GCACCTTGTC GCCTTGCGTA 


 


TAATATTTGC CCATGGGGGT GGGCGAAGAA CTCCAGCATG


 


AGATCCCCGC GCTGGAGGAT GACTTAGCGG TCGCCGTAGT


 


CGTGGAACAG CGGAACGCAT ATTATAAACG GGTACCCCCA


 


CCCGCTTCTT GAGGTCGTAC TCTAGGGGCG CGACCTCCTA


 


CATCCAGCCG GCGTCCCGGA AAACGATTCC GAAGCCCAAC


 


CTTTCATAGA AGGCGGCGGT GGAATCGAAA TCTCGTGATG


 


GCAGGTTGGG CGTCGCTTGG GTAGGTCGGC CGCAGGGCCT


 


TTTGCTAAGG CTTCGGGTTG GAAAGTATCT TCCGCCGCCA


 


CCTTAGCTTT AGAGCACTAC CGTCCAACCC GCAGCGAACC


 


TCGGTCATTT CGAACCCCAG AGTCCCGCTC AGAAGAACTC


 


GTCAAGAAGG CGATAGAAGG CGATGCGCTG CGAATCGGGA


 


GCGGCGATAC CGTAAAGCAC AGCCAGTAAA GCTTGGGGTC


 


TCAGGGCGAG TCTTCTTGAG CAGTTCTTCC GCTATCTTCC


 


GCTACGCGAC GCTTAGCCCT CGCCGCTATG GCATTTCGTG


 


GAGGAAGCGG TCAGCCCATT CGCCGCCAAG CTCTTCAGCA


 


ATATCACGGG TAGCCAACGC TATGTCCTGA TAGCGGTCCG


 


CCACACCCAG CCGGCCACAG CTCCTTCGCC AGTCGGGTAA


 


GCGGCGGTTC GAGAAGTCGT TATAGTGCCC ATCGGTTGCG


 


ATACAGGACT ATCGCCAGGC GGTGTGGGTC GGCCGGTGTC


 


TCGATGAATC CAGAAAAGCG GCCATTTTCC ACCATGATAT


 


TCGGCAAGCA GGCATCGCCA TGGGTCACGA CGAGATCCTC


 


GCCGTCGGGC ATGCGCGCCT AGCTACTTAG GTCTTTTCGC


 


CGGTAAAAGG TGGTACTATA AGCCGTTCGT CCGTAGCGGT


 


ACCCAGTGCT GCTCTAGGAG CGGCAGCCCG TACGCGCGGA


 


TGAGCCTGGC GAACAGTTCG GCTGGCGCGA GCCCCTGATG


 


CTCTTCGTCC AGATCATCCT GATCGACAAG ACCGGCTTCC


 


ATCCGAGTAC GTGCTCGCTC ACTCGGACCG CTTGTCAAGC


 


CGACCGCGCT CGGGGACTAC GAGAAGCAGG TCTAGTAGGA


 


CTAGCTGTTC TGGCCGAAGG TAGGCTCATG CACGAGCGAG


 


GATGCGATGT TTCGCTTGGT GGTCGAATGG GCAGGTAGCC


 


GGATCAAGCG TATGCAGCCG CCGCATTGCA TCAGCCATGA


 


TGGATACTTT CTCGGCAGGA CTACGCTACA AAGCGAACCA


 


CCAGCTTACC CGTCCATCGG CCTAGTTCGC ATACGTCGGC


 


GGCGTAACGT AGTCGGTACT ACCTATGAAA GAGCCGTCCT


 


GCAAGGTGAG ATGACAGGAG ATCCTGCCCC GGCACTTCGC


 


CCAATAGCAG CCAGTCCCTT CCCGCTTCAG TGACAACGTC


 


GAGCACAGCT GCGCAAGGAA CGTTCCACTC TACTGTCCTC


 


TAGGACGGGG CCGTGAAGCG GGTTATCGTC GGTCAGGGAA


 


GGGCGAAGTC ACTGTTGCAG CTCGTGTCGA CGCGTTCCTT


 


CGCCCGTCGT GGCCAGCCAC GATAGCCGCG CTGCCTCGTC


 


CTGCAGTTCA TTCAGGGCAC CGGACAGGTC GGTCTTGACA


 


AAAAGAACCG GGCGCCCCTG GCGGGCAGCA CCGGTCGGTG


 


CTATCGGCGC GACGGAGCAG GACGTCAAGT AAGTCCCGTG


 


GCCTGTCCAG CCAGAACTGT TTTTCTTGGC CCGCGGGGAC


 


CGCTGACAGC CGGAACACGG CGGCATCAGA GCAGCCGATT


 


GTCTGTTGTG CCCAGTCATA GCCGAATAGC CTCTCCACCC


 


AAGCGGCCGG AGAACCTGCG GCGACTGTCG GCCTTGTGCC


 


GCCGTAGTCT CGTCGGCTAA CAGACAACAC GGGTCAGTAT


 


CGGCTTATCG GAGAGGTGGG TTCGCCGGCC TCTTGGACGC


 


TGCAATCCAT CTTGTTCAAT CATGCGAAAC GATCCTCATC


 


CTGTCTCTTG ATCAGATCTT GATCCCCTGC GCCATCAGAT


 


CCTTGGCGGC AAGAAAGCCA ACGTTAGGTA GAACAAGTTA


 


GTACGCTTTG CTAGGAGTAG GACAGAGAAC TAGTCTAGAA


 


CTAGGGGACG CGGTAGTCTA GGAACCGCCG TTCTTTCGGT


 


TCCAGTTTAC TTTGCAGGGC TTCCCAACCT TACCAGAGGG


 


CGCCCCAGCT GGCAATTCCG GTTCGCTTGC TGTCCATAAA


 


ACCGCCCAGT CTAGCTATCG AGGTCAAATG AAACGTCCCG


 


AAGGGTTGGA ATGGTCTCCC GCGGGGTCGA CCGTTAAGGC


 


CAAGCGAACG ACAGGTATTT TGGCGGGTCA GATCGATAGC


 


CCATGTAAGC CCACTGCAAG CTACCTGCTT TCTCTTTGCG


 


CTTGCGTTTT CCCTTGTCCA GATAGCCCAG TAGCTGACAT


 


TCATCCCAGG TGGCACTTTT GGTACATTCG GGTGACGTTC


 


GATGGACGAA AGAGAAACGC GAACGCAAAA GGGAACAGGT


 


CTATCGGGTC ATCGACTGTA AGTAGGGTCC ACCGTGAAAA


 


CGGGGAAATG TGCGCGCCCG CGTTCCTGCT GGCGCTGGGC


 


CTGTTTCTGG CGCTGGACTT CCCGCTGTTC CGTCAGCAGC


 


TTTTCGCCCA CGGCCTTGAT GCCCCTTTAC ACGCGCGGGC


 


GCAAGGACGA CCGCGACCCG GACAAAGACC GCGACCTGAA


 


GGGCGACAAG GCAGTCGTCG AAAAGCGGGT GCCGGAACTA


 


GATCGCGGCG GCCTTGGCCT GCATATCCCG ATTCAACGGC


 


CCCAGGGCGT CCAGAACGGG CTTCAGGCGC TCCCGAAGGT


 


CTCGGGCCGT CTCTTGGGCT CTAGCGCCGC CGGAACCGGA


 


CGTATAGGGC TAAGTTGCCG GGGTCCCGCA GGTCTTGCCC


 


GAAGTCCGCG AGGGCTTCCA GAGCCCGGCA GAGAACCCGA


 


TGATCGGCCT TCTTGCGCAT CTCACGCGCT CCTGCGGCGG


 


CCTGTAGGGC AGGCTCATAC CCCTGCCGAA CCGCTTTTGT


 


CAGCCGGTCG GCCACGGCTT ACTAGCCGGA AGAACGCGTA


 


GAGTGCGCGA GGACGCCGCC GGACATCCCG TCCGAGTATG


 


GGGACGGCTT GGCGAAAACA GTCGGCCAGC CGGTGCCGAA


 


CCGGCGTCTC AACGCGCTTT GAGATTCCCA GCTTTTCGGC


 


CAATCCCTGC GGTGCATAGG CGCGTGGCTC GACCGCTTGC


 


GGGCTGATGG TGACGTGGCC GGCCGCAGAG TTGCGCGAAA


 


CTCTAAGGGT CGAAAAGCCG GTTAGGGACG CCACGTATCC


 


GCGCACCGAG CTGGCGAACG CCCGACTACC ACTGCACCGG


 


CACTGGTGGC CGCTCCAGGG CCTCGTAGAA CGCCTGAATG


 


CGCGTGTGAC GTGCCTTGCT GCCCTCGATG CCCCGTTGCA


 


GCCCTAGATC GGCCACAGCG GTGACCACCG GCGAGGTCCC


 


GGAGCATCTT GCGGACTTAC GCGCACACTG CACGGAACGA


 


CGGGAGCTAC GGGGCAACGT CGGGATCTAG CCGGTGTCGC


 


GCCGCAAACG TGGTCTGGTC GCGGGTCATC TGCGCTTTGT


 


TGCCGATGAA CTCCTTGGCC GACAGCCTGC CGTCCTGCGT


 


CAGCGGCACC ACGAACGCGG CGGCGTTTGC ACCAGACCAG


 


CGCCCAGTAG ACGCGAAACA ACGGCTACTT GAGGAACCGG


 


CTGTCGGACG GCAGGACGCA GTCGCCGTGG TGCTTGCGCC


 


TCATGTGCGG GCTGGTTTCG TCACGGTGGA TGCTGGCCGT


 


CACGATGCGA TCCGCCCCGT ACTTGTCCGC CAGCCACTTG


 


TGCGCCTTCT CGAAGAACGC AGTACACGCC CGACCAAAGC


 


AGTGCCACCT ACGACCGGCA GTGCTACGCT AGGCGGGGCA


 


TGAACAGGCG GTCGGTGAAC ACGCGGAAGA GCTTCTTGCG


 


CGCCTGCTGT TCTTGGCTGG CCGACTTCCA CCATTCCGGG


 


CTGGCCGTCA TGACGTACTC GACCGCCAAC ACAGCGTCCT


 


TGCGCCGCTT CTCTGGCAGC GCGGACGACA AGAACCGACC


 


GGCTGAAGGT GGTAAGGCCC GACCGGCAGT ACTGCATGAG


 


CTGGCGGTTG TGTCGCAGGA ACGCGGCGAA GAGACCGTCG


 


AACTCGCGCA GTCGGCCCAT CGCTTCATCG GTGCTGCTGG


 


CCGCCCAGTG CTCGTTCTCT GGCGTCCTGC TGGCGTCAGC


 


GTTGGGCGTC TCGCGCTCGC TTGAGCGCGT CAGCCGGGTA


 


GCGAAGTAGC CACGACGACC GGCGGGTCAC GAGCAAGAGA


 


CCGCAGGACG ACCGCAGTCG CAACCCGCAG AGCGCGAGCG


 


GGTAGGCGTG CTTGAGACTG GCCGCCACGT TGCCCATTTT


 


CGCCAGCTTC TTGCATCGCA TGATCGCGTA TGCCGCCATG


 


CCTGCCCCTC CCTTTTGGTG CCATCCGCAC GAACTCTGAC


 


CGGCGGTGCA ACGGGTAAAA GCGGTCGAAG AACGTAGCGT


 


ACTAGCGCAT ACGGCGGTAC GGACGGGGAG GGAAAACCAC


 


TCCAACCGGC TCGACGGGGG CAGCGCAAGG CGGTGCCTCC


 


GGCGGGCCAC TCAATGCTTG AGTATACTCA CTAGACTTTG


 


CTTCGCAAAG TCGTGACCGC AGGTTGGCCG AGCTGCCCCC


 


GTCGCGTTCC GCCACGGAGG CCGCCCGGTG AGTTACGAAC


 


TCATATGAGT GATCTGAAAC GAAGCGTTTC AGCACTGGCG


 


CTACGGCGGC TGCGGCGCCC TACGGGCTTG CTCTCCGGGC


 


TTCGCCCTGC GCGGTCGCTG CGCTCCCTTG CCAGCCCGTG


 


GATATGTGGA CGATGGCCGC GATGCCGCCG ACGCCGCGGG


 


ATGCCCGAAC GAGAGGCCCG AAGCGGGACG CGCCAGCGAC


 


GCGAGGGAAC GGTCGGGCAC CTATACACCT GCTACCGGCG


 


GAGCGGCCAC CGGCTGGCTC GCTTCGCTCG GCCCGTGGAC


 


AACCCTGCTG GACAAGCTGA TGGACAGGCT GCGCCTGCCC


 


ACGAGCTTGA CCACAGGGAT CTCGCCGGTG GCCGACCGAG


 


CGAAGCGAGC CGGGCACCTG TTGGGACGAC CTGTTCGACT


 


ACCTGTCCGA CGCGGACGGG TGCTCGAACT GGTGTCCCTA


 


TGCCCACCGG CTACCCAGCC TTCGACCACA TACCCACCGG


 


CTCCAACTGC GCGGCCTGCG GCCTTGCCCC ATCAATTTTT


 


TTAATTTTCT CTGGGGAAAA ACGGGTGGCC GATGGGTCGG


 


AAGCTGGTGT ATGGGTGGCC GAGGTTGACG CGCCGGACGC


 


CGGAACGGGG TAGTTAAAAA AATTAAAAGA GACCCCTTTT


 


GCCTCCGGCC TGCGGCCTGC GCGCTTCGCT TGCCGGTTGG


 


ACACCAAGTG GAAGGCGGGT CAAGGCTCGC GCAGCGACCG


 


CGCAGCGGCT TGGCCTTGAC CGGAGGCCGG ACGCCGGACG


 


CGCGAAGCGA ACGGCCAACC TGTGGTTCAC CTTCCGCCCA


 


GTTCCGAGCG CGTCGCTGGC GCGTCGCCGA ACCGGAACTG


 


GCGCCTGGAA CGACCCAAGC CTATGCGAGT GGGGGCAGTC


 


GAAGGCGAAG CCCGCCCGCC TGCCCCCCGA GCCTCACGGC


 


GGCGAGTGCG GGGGTTCCAA CGCGGACCTT GCTGGGTTCG


 


GATACGCTCA CCCCCGTCAG CTTCCGCTTC GGGCGGGCGG


 


ACGGGGGGCT CGGAGTGCCG CCGCTCACGC CCCCAAGGTT


 


GGGGGCAGCG CCACCTTGGG CAAGGCCGAA GGCCGCGCAG


 


TCGATCAACA AGCCCCGGAG GGGCCACTTT TTGCCGGAG


 


CCCCCGTCGC GGTGGAACCC GTTCCGGCTT CCGGCGCGTC


 


AGCTAGTTGT TCGGGGCCTC CCCGGTGAAA AACGGCCTC


 


SEQ ID: 05


MPELAVRTEF DYSSEIYKDA YSRINAIVIE GEQEAYSNYL


 


QMAELLPEDK EELTRLAKME NRHKKGFQAC GNNLQVNPDM


 


PYAQEFFAGL HGNFQHAFSE GKVVTCLLIQ ALIIEAFAIA


 


AYNIYIPVAD DFARKITEGV VKDEYTHLNY GEEWLKANFA


 


TAKEELEQAN KENLPLVWKM LNQVQGDAKV LGMEKEALVE


 


DFMISYGEAL SNIGFSTREI MRMSSYGLAG V


 


SEQ ID: 06


MFGLIGHLTS LEHAQAVAED LGYPEYANQG LDFWCSAPPQ


 


VVDNFQVKSV TGQVIEGKYV ESCFLPEMLT QRRIKAAIRK


 


ILNAMALAQK VGLDITALGG FSSIVFEEFN LKQNNQVRNV


 


ELDFQRFTTG NTHTAYVICR QVESGAKQLG IDLSQATVAV


 


CGATGDIGSA VCRWLDSKHQ VKELLLIARN RQRLENLQEE


 


LGRGKIMDLE TALPQADIIV WVASMPKGVE IAGEMLKKPC


 


LIVDGGYPKN LDTRVKADGV HILKGGIVEH SLDITWEIMK


 


IVEMDIPSRQ MFACFAEAIL LEFEGWRTNF SWGRNQISVN


 


KMEAIGEASV KHGFCPLVAL


 


SEQ ID: 07


CAGTCAATGG AGAGCATTGC CATAAGTAAA GGCATCCCCT


 


GCGTGATAAG ATTACCTTCA GAAAACAGAT AGTTGCTGGG


 


TTATCGCAGA TTTTTCTCGC GTCAGTTACC TCTCGTAACG


 


GTATTCATTT CCGTAGGGGA CGCACTATTC TAATGGAAGT


 


CTTTTGTCTA TCAACGACCC AATAGCGTCT AAAAAGAGCG


 


AACCAAATAA CTGTAAATAA TAACTGTCTC TGGGGCGACG


 


GTAGGCTTTA TATTGCCAAA TTTCGCCCGT GGGAGAAAGC


 


TAGGCTATTC AATGTTTATG TTGGTTTATT GACATTTATT


 


ATTGACAGAG ACCCCGCTGC CATCCGAAAT ATAACGGTTT


 


AAAGCGGGCA CCCTCTTTCG ATCCGATAAG TTACAAATAC


 


GAGGACTCCT


 


SEQ ID: 08


CCTGGCTCAG GACGAACGCT GGCGGCGTGC TTAACACATG


 


CAAGTCGAGC GGTAAGGCCC TTCGGGGTAC ACGAGCGGCG


 


AACGGGTGAG TAACACGTGG GGACCGAGTC CTGCTTGCGA


 


CCGCCGCACG AATTGTGTAC GTTCAGCTCG CCATTCCGGG


 


AAGCCCCATG TGCTCGCCGC TTGCCCACTC ATTGTGCACC


 


GTGATCTGCC CTGCACTTCG GGATAAGCCT GGGAAACTGG


 


GTCTAATACC GGATATGACC TTCGGCTGCA TGGCTGAGGG


 


TGGAAAGGTT TACTGGTGCA CACTAGACGG GACGTGAAGC


 


CCTATTCGGA CCCTTTGACC CAGATTATGG CCTATACTGG


 


AAGCCGACGT ACCGACTCCC ACCTTTCCAA ATGACCACGT


 


GGATGGGCCC GCGGCCTATC AGCTTGTTGG TGGGGTAATG


 


GCCTACCAAG GCGACGACGG GTAGCCGACC TGAGAGGGTG


 


ACCGGCCACA CTGGGACTGA CCTACCCGGG CGCCGGATAG


 


TCGAACAACC ACCCCATTAC CGGATGGTTC CGCTGCTGCC


 


CATCGGCTGG ACTCTCCCAC TGGCCGGTGT GACCCTGACT


 


GACACGGCCC AGACTCCTAC GGGAGGCAGC AGTGGGGAAT


 


ATTGCACAAT GGGCGAAAGC CTGATGCAGC GACGCCGCGT


 


GAGGGATGAC GGCCTTCGGG CTGTGCCGGG TCTGAGGATG


 


CCCTCCGTCG TCACCCCTTA TAACGTGTTA CCCGCTTTCG


 


GACTACGTCG CTGCGGCGCA CTCCCTACTG CCGGAAGCCC


 


TTGTAAACCT CTTTCAGCAG GGACGAAGCG AAAGTGACGG


 


TACCTGCAGA AGAAGCACCG GCCAACTACG TGCCAGCAGC


 


CGCGGTAATA CGTAGGGTGC AACATTTGGA GAAAGTCGTC


 


CCTGCTTCGC TTTCACTGCC ATGGACGTCT TCTTCGTGGC


 


CGGTTGATGC ACGGTCGTCG GCGCCATTAT GCATCCCACG


 


AAGCGTTGTC CGGAATTACT GGGCGTAAAG AGCTCGTAGG


 


CGGTTTGTCG CGTCGTCTGT GAAAACTCAN AGCTCAACCT


 


CGAGCTTGCA GGCGATACGG TTCGCAACAG GCCTTAATGA


 


CCCGCATTTC TCGAGCATCC GCCAAACAGC GCAGCAGACA


 


CTTTTGAGTN TCGAGTTGGA GCTCGAACGT CCGCTATGCC


 


GCAGACTTGA GTACTGCAGG GGAGACTGGA ATTCCTGGTG


 


TAGCGGTGAA ATGCGCAGAT ATCAGGAGGA ACACCGGTGG


 


CGAAGGCGGG TCTCTGGGCA CGTCTGAACT CATGACGTCC


 


CCTCTGACCT TAAGGACCAC ATCGCCACTT TACGCGTCTA


 


TAGTCCTCCT TGTGGCCACC GCTTCCGCCC AGAGACCCGT


 


GTAACTGACG CTGAGGAGCG AAAGCGTGGG TAGCAAACAG


 


GATTAGATAC CCTGGTAGTC CACGCCGTAA ACGGTGGGCG


 


CTAGGTGTGG GTTTCCTTCC CATTGACTGC GACTCCTCGC


 


TTTCGCACCC ATCGTTTGTC CTAATCTATG GGACCATCAG


 


GTGCGGCATT TGCCACCCGC GATCCACACC CAAAGGAAGG


 


ACGGGATCCG TGCCGTAGTT AACGCATTAA GCGCCCCGCC


 


TGGGGAGTAC GGCCGCAAGG TTAAAACTCA AAGGAATTGA


 


CGGGGGCCCG CACAAGCGGC TGCCCTAGGC ACGGCATCAA


 


TTGCGTAATT CGCGGGGCGG ACCCCTCATG CCGGCGTTCC


 


AATTTTGAGT TTCCTTAACT GCCCCCGGGC GTGTTCGCCG


 


GGAGCATGTG GATTAATTCG ATGCAACGCG AAGAACCTTA


 


CCTGGGTTTG ACATATACCG GAAAGCCGTA GAGATACCGC


 


CCCCCTTGTG GTCGGTATAC CCTCGTACAC CTAATTAAGC


 


TACGTTGCGC TTCTTGGAAT GGACCCAAAC TGTATATGGC


 


CTTTCGGCAT CTCTATGGCG GGGGGAACAC CAGCCATATG


 


AGGTGGTGCA TGGCTGTCGT CAGCTCGTGT CGTGAGATGT


 


TGGGTTAAGT CCCGCAACGA GCGCAACCCT TGTCTTATGT


 


TGCCAGCACG TAATGGTGGG TCCACCACGT ACCGACAGCA


 


GTCGAGCACA GCACTCTACA ACCCAATTCA GGGCGTTGCT


 


CGCGTTGGGA ACAGAATACA ACGGTCGTGC ATTACCACCC


 


GACTCGTAAG AGACTGCCGG GGTCAACTCG GAGGAAGGTG


 


GGGACGACGT CAAGTCATCA TGCCCCTTAT GTCCAGGGCT


 


TCACACATGC TACAATGGCC CTGAGCATTC TCTGACGGCC


 


CCAGTTGAGC CTCCTTCCAC CCCTGCTGCA GTTCAGTAGT


 


ACGGGGAATA CAGGTCCCGA AGTGTGTACG ATGTTACCGG


 


GGTACAGAGG GCTGCGATAC CGTGAGGTGG AGCGAATCCC


 


TTAAAGCCGG TCTCAGTTCG GATCGGGGTC TGCAACTCGA


 


CCCCGTGAAG TCGGAGTCGC CCATGTCTCC CGACGCTATG


 


GCACTCCACC TCGCTTAGGG AATTTCGGCC AGAGTCAAGC


 


CTAGCCCCAG ACGTTGAGCT GGGGCACTTC AGCCTCAGCG


 


TAGTAATCGC AGATCAGCAA CGCTGCGGTG AATACGTTCC


 


CGGGCCTTGT ACACACCGCC CGTCACGTCA TGAAAGTCGG


 


TAACACCCGA AGCCGGTGGC ATCATTAGCG TCTAGTCGTT


 


GCGACGCCAC TTATGCAAGG GCCCGGAACA TGTGTGGCGG


 


GCAGTGCAGT ACTTTCAGCC ATTGTGGGCT TCGGCCACCG


 


CTAACCCCTT GTGGGAGGGA GCCGTCGAAG GTGGGATCGG


 


CGATTGGGAC GAAGTCGTAA CAAGGTAGCC GTACCGGAAG


 


GGATTGGGGA ACACCCTCCC TCGGCAGCTT CCACCCTAGC


 


CGCTAACCCT GCTTCAGCAT TGTTCCATCG GCATGGCCTT


 


CC


 


SEQ ID: 09


TCAACGGAGA GTTTGATCCT GGCTCAGGAC GAACGCTGGC


 


GGCGTGCTTA ACACATGCAA GTCGAGCGGT AAGGCCCTTC


 


GGGGTACACG AGCGGCGAAC AGTTGCCTCT CAAACTAGGA


 


CCGAGTCCTG CTTGCGACCG CCGCACGAAT TGTGTACGTT


 


CAGCTCGCCA TTCCGGGAAG CCCCATGTGC TCGCCGCTTG


 


GGGTGAGTAA CACGTGGGTG ATCTGCCCTG CACTTCGGGA


 


TAAGCCTGGG AAACTGGGTC TAATACCGGA TATGACCTTC


 


GGCTGCATGG CCGTTGGTGG CCCACTCATT GTGCACCCAC


 


TAGACGGGAC GTGAAGCCCT ATTCGGACCC TTTGACCCAG


 


ATTATGGCCT ATACTGGAAG CCGACGTACC GGCAACCACC


 


AAAGGTTTAC TGGTGCAGGA TGGGCCCGCG GCCTATCAGC


 


TTGTTGGTGG GGTAATGGCC TACCAAGGCG ACGACGGGTA


 


GCCGACCTGA GAGGGTGACC TTTCCAAATG ACCACGTCCT


 


ACCCGGGCGC CGGATAGTCG AACAACCACC CCATTACCGG


 


ATGGTTCCGC TGCTGCCCAT CGGCTGGACT CTCCCACTGG


 


GGCCACACTG GGACTGAGAC ACGGCCCAGA CTCCTACGGG


 


AGGCAGCAGT GGGGAATATT GCACAATGGG CGAAAGCCTG


 


ATGCAGCGAC GCCGCGTGAG CCGGTGTGAC CCTGACTCTG


 


TGCCGGGTCT GAGGATGCCC TCCGTCGTCA CCCCTTATAA


 


CGTGTTACCC GCTTTCGGAC TACGTCGCTG CGGCGCACTC


 


GGATGACGGC CTTCGGGTTG TAAACCTCTT TCAGCAGGGA


 


CGAAGCGAAA GTGACGGTAC CTGCAGAAGA AGCACCGGCC


 


AACTACGTGC CAGCAGCCGC CCTACTGCCG GAAGCCCAAC


 


ATTTGGAGAA AGTCGTCCCT GCTTCGCTTT CACTGCCATG


 


GACGTCTTCT TCGTGGCCGG TTGATGCACG GTCGTCGGCG


 


GGTAATACGT AGGGTGCAAG CGTTGTCCGG AATTACTGGG


 


CGTAAAGAGC TCGTAGGCGG TTTGTCGCGT CGTCTGTGAA


 


AACTCGAGGC TCAACCTCGA CCATTATGCA TCCCACGTTC


 


GCAACAGGCC TTAATGACCC GCATTTCTCG AGCATCCGCC


 


AAACAGCGCA GCAGACACTT TTGAGCTCCG AGTTGGAGCT


 


GCTTGCAGGC GATACGGGCA GACTTGAGTA CTGCAGGGGA


 


GACTGGAATT CCTGGTGTAG CGGTGAAATG CGCAGATATC


 


AGGAGGAACA CCGGTGGCGA CGAACGTCCG CTATGCCCGT


 


CTGAACTCAT GACGTCCCCT CTGACCTTAA GGACCACATC


 


GCCACTTTAC GCGTCTATAG TCCTCCTTGT GGCCACCGCT


 


AGGCGGGTCT CTGGGCAGTA ACTGACGCTG AGGAGCGAAA


 


GCGTGGGTAG CGAACAGGAT TAGATACCCT GGTAGTCCAC


 


GCCGTAAACG GTGGGCGCTA TCCGCCCAGA GACCCGTCAT


 


TGACTGCGAC TCCTCGCTTT CGCACCCATC GCTTGTCCTA


 


ATCTATGGGA CCATCAGGTG CGGCATTTGC CACCCGCGAT


 


GGTGTGGGTT TCCTTCCACG GGATCCGTGC CGTAGCTAAC


 


GCATTAAGCG CCCCGCCTGG GGAGTACGGC CGCAAGGCTA


 


AAACTCAAAG GAATTGACGG CCACACCCAA AGGAAGGTGC


 


CCTAGGCACG GCATCGATTG CGTAATTCGC GGGGCGGACC


 


CCTCATGCCG GCGTTCCGAT TTTGAGTTTC CTTAACTGCC


 


GGGCCCGCAC AAGCGGCGGA GCATGTGGAT TAATTCGATG


 


CAACGCGAAG AACCTTACCT GGGTTTGACA TATACCGGAA


 


AGCTGCAGAG ATGTGGCCCC CCCGGGCGTG TTCGCCGCCT


 


CGTACACCTA ATTAAGCTAC GTTGCGCTTC TTGGAATGGA


 


CCCAAACTGT ATATGGCCTT TCGACGTCTC TACACCGGGG


 


CCTTGTGGTC GGTATACAGG TGGTGCATGG CTGTCGTCAG


 


CTCGTGTCGT GAGATGTTGG GTTAAGTCCC GCAACGAGCG


 


CAACCCTTGT CTTATGTTGC GGAACACCAG CCATATGTCC


 


ACCACGTACC GACAGCAGTC GAGCACAGCA CTCTACAACC


 


CAATTCAGGG CGTTGCTCGC GTTGGGAACA GAATACAACG


 


CAGCACGTAA TGGTGGGGAC TCGTAAGAGA CTGCCGGGGT


 


CAACTCGGAG GAAGGTGGGG ACGACGTCAA GTCATCATGC


 


CCCTTATGTC CAGGGCTTCA GTCGTGCATT ACCACCCCTG


 


AGCATTCTCT GACGGCCCCA GTTGAGCCTC CTTCCACCCC


 


TGCTGCAGTT CAGTAGTACG GGGAATACAG GTCCCGAAGT


 


CACATGCTAC AATGGCCGGT ACAGAGGGCT GCGATACCGT


 


GAGGTGGAGC GAATCCCTTA AAGCCGGTCT CAGTTCGGAT


 


CGGGGTCTGC AACTCGACCC GTGTACGATG TTACCGGCCA


 


TGTCTCCCGA CGCTATGGCA CTCCACCTCG CTTAGGGAAT


 


TTCGGCCAGA GTCAAGCCTA GCCCCAGACG TTGAGCTGGG


 


CGTGAAGTCG GAGTCGCTAG TAATCGCAGA TCAGCAACGC


 


TGCGGTGAAT ACGTTCCCGG GCCTTGTACA CACCGCCCGT


 


CACGTCATGA AAGTCGGTAA GCACTTCAGC CTCAGCGATC


 


ATTAGCGTCT AGTCGTTGCG ACGCCACTTA TGCAAGGGCC


 


CGGAACATGT GTGGCGGGCA GTGCAGTACT TTCAGCCATT


 


CACCCGAAGC CGGTGGCCTA ACCCCTCGTG GGAGGGAGCC


 


GTCGAAGGTG GGATCGGCGA TTGGGACGAA GTCGTAACAA


 


GGTAGCCGTA CCGGAAGGTG GTGGGCTTCG GCCACCGGAT


 


TGGGGAGCAC CCTCCCTCGG CAGCTTCCAC CCTAGCCGCT


 


AACCCTGCTT CAGCATTGTT CCATCGGCAT GGCCTTCCAC


 


CGGCTGGATC ACCTCCTTTC TGCCGACCTA GTGGAGGAAA GA


 


SEQ ID: 10


ACGTGGCGGC ATGCCTTACA CATGCAAGTC GAACGGCAGC


 


GCGGACTTCG GTCTGGCGGC GAGTGGCGAA CGGGTGAGTA


 


ATACATCGGA ACGTACCCTG TGCACCGCCG TACGGAATGT


 


GTACGTTCAG CTTGCCGTCG CGCCTGAAGC CAGACCGCCG


 


CTCACCGCTT GCCCACTCAT TATGTAGCCT TGCATGGGAC


 


TTGTGGGGGA TAACTAGTCG AAAGATTAGC TAATACCGCA


 


TACGACCTGA GGGTGAAAGT GGGGGACCGC AAGGCCTCAC


 


GCAGCAGGAG CGGCCGATGT AACACCCCCT ATTGATCAGC


 


TTTCTAATCG ATTATGGCGT ATGCTGGACT CCCACTTTCA


 


CCCCCTGGCG TTCCGGAGTG CGTCGTCCTC GCCGGCTACA


 


CTGATTAGCT AGTTGGTGGG GTAAAGGCCC ACCAAGGCGA


 


CGATCAGTAG CTGGTCTGAG AGGACGATCA GCCACACTGG


 


GACTGAGACA CGGCCCAGAC GACTAATCGA TCAACCACCC


 


CATTTCCGGG TGGTTCCGCT GCTAGTCATC GACCAGACTC


 


TCCTGCTAGT CGGTGTGACC CTGACTCTGT GCCGGGTCTG


 


TCCTACGGGA GGCAGCAGTG GGGAATTTTG GACAATGGGG


 


GCAACCCTGA TCCAGCAATG CCGCGTGTGT GAAGAAGGCC


 


TTCGGGTTGT AAAGCACTTT AGGATGCCCT CCGTCGTCAC


 


CCCTTAAAAC CTGTTACCCC CGTTGGGACT AGGTCGTTAC


 


GGCGCACACA CTTCTTCCGG AAGCCCAACA TTTCGTGAAA


 


TGTCCGGAAA GAAATCGCGC TGGTTAATAC CTGCGTGATG


 


ACGGTACCGG AAGAATAAGC ACCGGCTAAC TACGTGCCAG


 


CAGCCGCGGT AATACGTAGG ACAGGCCTTT CTTTAGCGCG


 


ACCAATTATG GACGCACTAC TGCCATGGCC TTCTTATTCG


 


TGGCCGATTG ATGCACGGTC GTCGGCGCCA TTATGCATCC


 


GTGCGAGCGT TAATCGGAAT TACTGGGCGT AAAGCGTGCG


 


CAGGCGGTTT TGTAAGACAG GCGTGAAATC CCCGGGCTTA


 


ACCTGGGAAT TGCGCTTGTG CACGCTCGCA ATTAGCCTTA


 


ATGACCCGCA TTTCGCACGC GTCCGCCAAA ACATTCTGTC


 


CGCACTTTAG GGGCCCGAAT TGGACCCTTA ACGCGAACAC


 


ACTGCAAGGC TAGAGTGCGT CAGAGGGGGG TAGAATTCCA


 


CGTGTAGCAG TGAAATGCGT AGAGATGTGG AGGAATACCG


 


ATGGCGAAGG CGAGCCCCCT TGACGTTCCG ATCTCACGCA


 


GTCTCCCCCC ATCTTAAGGT GCACATCGTC ACTTTACGCA


 


TCTCTACACC TCCTTATGGC TACCGCTTCC GCTCGGGGGA


 


GGACCTTGAC TGACGCTCAT GCACGAAAGC GTGGGGAGCA


 


AACAGGATTA GATACCCTGG TAGTCCACGC CCTAAACGAT


 


GTCAACTAGT TGTTGGGATT CCTGGAACTG ACTGCGAGTA


 


CGTGCTTTCG CACCCCTCGT TTGTCCTAAT CTATGGGACC


 


ATCAGGTGCG GGATTTGCTA CAGTTGATCA ACAACCCTAA


 


CATTTTCTCA GTAACGTAGC TAACGCGTGA AGTTGACCGC


 


CTGGGGAGTA CGGCTGCAAG ATTAAAACTC AAAGGAATTG


 


ACGGGGACCC GCACAAGCGG GTAAAAGAGT CATTGCATCG


 


ATTGCGCACT TCAACTGGCG GACCCCTCAT GCCGACGTTC


 


TAATTTTGAG TTTCCTTAAC TGCCCCTGGG CGTGTTCGCC


 


TGGATGATGT GGATTAATTC GATGCAACGC GAAAAACCTT


 


ACCTACCCTT GACATGCCCT AACGAAGCAG AGATGCATTA


 


GTGCCCGCAA AGGGAAAGTG ACCTACTACA CCTAATTAAG


 


CTACGTTGCG CTTTTTGGAA TGGATGGGAA CTGTACGGGA


 


TTGCTTCGTC TCTACGTAAT CACGGGCGTT TCCCTTTCAC


 


GGACACAGGT GCTGCATGGC TGTCGTCAGC TCGTGTCGTG


 


AGATGTTGGG TTAAGTCCCG CAACGAGCGC AACCCTTGTC


 


TCTAGTTGCC TACGCAAGAG CCTGTGTCCA CGACGTACCG


 


ACAGCAGTCG AGCACAGCAC TCTACAACCC AATTCAGGGC


 


GTTGCTCGCG TTGGGAACAG AGATCAACGG ATGCGTTCTC


 


CACTCTAGAG AGACTGCCGG TGACAAACCG GAGGAAGGTG


 


GGGATGACGT CAAGTCCTCA TGGCCCTTAT GGGTAGGGCT


 


TCACACGTCA TACAATGGTG GTGAGATCTC TCTGACGGCC


 


ACTGTTTGGC CTCCTTCCAC CCCTACTGCA GTTCAGGAGT


 


ACCGGGAATA CCCATCCCGA AGTGTGCAGT ATGTTACCAC


 


CGTACAGAGG GTTGCCAACC CGCGAGGGGG AGCTAATCCC


 


AGAAAACGCA TCGTAGTCCG GATCGTAGTC TGCAACTCGA


 


CTACGTGAAG CTGGAATCGC GCATGTCTCC CAACGGTTGG


 


GCGCTCCCCC TCGATTAGGG TCTTTTGCGT AGCATCAGGC


 


CTAGCATCAG ACGTTGAGCT GATGCACTTC GACCTTAGCG


 


TAGTAATCGC GGATCAGCAT GCCGCGGTGA ATACGTTCCC


 


GGGTCTTGTA CACACCGCCC GTCACACCAT GGGAGTGGGT


 


TTTGCCAGAA GTAGTTAGCC ATCATTAGCG CCTAGTCGTA


 


CGGCGCCACT TATGCAAGGG CCCAGAACAT GTGTGGCGGG


 


CAGTGTGGTA CCCTCACCCA AAACGGTCTT CATCAATCGG


 


TAACCGCAAG GAGGGCGATT ACCACGGCAG GGTTCATGAC


 


TGGGGTGAAG TCGTAACAAG GTATTGGCGT TCCTCCCGCT


 


AATGGTGCCG TCCCAAGTAC TGACCCCACT TCAGCATTGT TCCA


 


SEQ ID: 11


MASIEDILEL EALEKDIFRG AVHPSVLKRT FGGQVAGQSL


 


VSAVRTVDER FEVHSLHGYF LRPGNPTEPT VYLVDRIRDG


 


RSFCTRRVTG IQDGKAIFTM SASFHSQDEG IEHQDTMPSV


 


PEPEELVDAQ TVEEMAATDL YREWKEWDVR IVPAGCTGKT


 


PGIAAKQRVW MRYRNKLPDD QVFHICTLAY LSDMTLLGAS


 


KVPHPGVVTQ TASLDHAMWF LRPFRADEWL LYDQTSPSAG


 


FGRALTQGRM FDRKGTMVAA VVQEGLTRIQ RDQDQRDIET GNMA






In some embodiments, the cell comprises a plasmid that contains one or more exogenous nucleic acid sequences encoding enzymes or proteins that include but are not limited to one or more of the following: an acyl carrier protein, a TE, a FAR, a FadR, a FAD, a fatty aldehyde reductase, a cytochrome P450 enzyme, a NADH or NADPH cytochrome P450 reductase, a desaturase, a hydroxylase, and an antibiotic resistance enabling protein; wherein the plasmid is at least 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% homologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
In one embodiment, the exogenous gene encodes a FadR. In some cases, the reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding aldehyde. In one embodiment, the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanal.
In some embodiments, the invention relates to a bacterial cell or a compositions comprising at least one bacterial cell that comprises at least a first and a second exogenous nucleic acid sequence, wherein the first nucleic acid sequence encodes a FadR or a functional fragment of a FadR and the second exogenous nucleic acid sequence encodes a fatty acyl-CoA ligase or a functional fragment thereof. In some embodiments, the functional fragments of the enzymes encoded by the one or more exogenous nucleic acid sequences are at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the nucleic acid sequences that encode the full-length amino acid sequence upon which the functional fragment is based. Any enzyme disclosed in this application and part of the invention may be replaced with a functional fragment. Any composition or cell disclosed in the application may be used in any disclosed method of this application.
In some embodiments, the genetic constructs contain sequences directing transcription and translation of the relevant exogenous (either heterologous or homologous) gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host. In some cells the exogenous gene is coding sequence and is in operable linkage with a promoter, and in some embodiments the promoter is derived from a gene endogenous to a species of the genus Rhodococcus or Ralstonia. Initiation control regions or promoters, which are useful to drive expression of the instant ORFs in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO; and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc as well as the amy, apr, npr promoters and various phage promoters useful for expression in the lipid-producing bacteria of the present invention. In other embodiments the promoter is upregulated in response to reduction or elimination of a cofactor in the culture media of the cell, such as at least a 3-fold upregulation as determined by transcript abundance in a cell when the cell is exposed to extracellular environment changes from containing at least 10 mM or 5 mM cofactor to containing no cofactor.
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, the genetic constructs of the present invention do not comprise a termination control region.
In some embodiments, the bacterial cell or the composition comprising the bacterial cell comprises at least one genetic construct, which comprises one or more coding sequences. In some embodiments, the invention relates to the bacterial cell or the composition comprising at least one bacterial cell wherein the at least one cell comprises two or more genetic constructs, three or more genetic constructs, or four or more genetic constructs, each comprising one or more coding sequences. In some embodiments, the coding sequences of the claimed invention encode at least one protein that modifies or accelerates lipid production in the host cell. In some embodiments the coding sequence encodes at least one protein that alters the levels of individual lipids or hydrocarbons produced by the cell as compared to the same cell not modified by an exogenous nucleic acid sequence. In some embodiments, the coding sequence may encode at least one protein that alters the amount of one specific lipid or hydrocarbon molecule of the cell as compared to the same cell not modified by the nucleic acid. For example, in one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes an increase in the ratio of C14:C16:C18 unsaturated lipids or desaturated hydrocarbons produced or secreted by the cell as compared to the C14:C16:C18 unsaturated lipids or desaturated hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of C14:C16:C18 unsaturated lipids or desaturated hydrocarbons produced or secreted by the cell as compared to the C14:C16:C18 unsaturated lipids or desaturated hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In some embodiments, the one or more cells comprising one or more exogenous nucleic acid sequences produces more hydrocarbon than the same one or more cells not transformed or modified with the one or more exogenous nucleic acid sequences.
In some embodiments, the bacterial cell produces and/or secretes one or more unsaturated lipids or hydrocarbons in a ratio greater than the ratio of unsaturated lipids or hydrocarbons produced and/or secreted by the same cell not comprising the one or more exogenous nucleic acid sequences. In some embodiments, the bacterial cell produces and/or secretes one or more unsaturated lipids or hydrocarbons, wherein at least 50% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 60% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 70% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 75% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms. In some embodiments, the bacterial cell produces and/or secretes one or more lipids or hydrocarbons, wherein at least 80% of the one or more lipids or hydrocarbons have 8 to 18 carbon atoms.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, wherein the one or more hydrocarbons have a carbon chain length of at least 8 carbon atoms. In some embodiments, The invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the one or more hydrocarbons comprise a mixture of hydrocarbon molecules having a carbon chain length from 8 carbon atoms to 18 carbon atoms. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the one or more lipids comprise a quantity of at least one alkene, or alkyne at a level higher than the quantity of the alkene, alkyne in the same microorganism not comprising the heterologous nucleic acid sequences. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 60% of one or more lipids by weight.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 65% of one or more lipids by weight. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 70% of one or more hydrocarbons by weight.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 75% of one or more lipids by weight. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 80% of one or more lipids by weight.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated lipids, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the microorganism produces and/or secretes at least 85% of one or more lipids by weight. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein less than 10% by weight of the hydrocarbons produced is methane. In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more organic compounds, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein less than 10% by weight of the organic compounds produced are organic acids with carbon chain length of four carbons or less.
In some embodiments, the invention relates to a composition comprising a microorganism that converts syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas into one or more unsaturated hydrocarbons, wherein the microorganism comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase; wherein the hydrocarbons produced comprise a mixture of at least two hydrocarbons having a carbon backbone from 8 to 18 carbon atoms.
The present invention also relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into lipids. In some embodiments, the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into lipid; wherein the cell produces and/or secretes at least 75% of one or more hydrocarbons by weight. In some embodiments, the invention elates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into lipid; wherein the cell produces and/or secretes at least 75% of one or more hydrocarbons by weight when cultured at least 42 degrees Celsius for at least 1 hour. In some embodiments, the bacterial cell is cultured without exposure to light.
In some embodiments, the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid acyl-ACP reductase and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into a hydrocarbon or mixture of hydrocarbons, and/or other lipids; wherein the cell is a strain of Rhodococcus opacus. 
In some embodiments, the invention relates to a bacterial cell comprising at least two exogenous nucleic acid sequences, wherein the at least two exogenous nucleic acid sequences encode fatty acid aldehyde acyl-ACP and fatty acid aldehyde decarbonylase, and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into a hydrocarbon or mixture of hydrocarbons, and/or other lipids; wherein the cell is a strain of Cupriavidus necator. 
In some embodiments, the invention relates to a bacterial cell comprising a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase; and wherein the cell converts gaseous CO2 and/or gaseous H2 and/or syngas into a lipid or mixture of lipids. In some embodiments, the bacterial cell comprises no more than eight exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than seven exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than six exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than four exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than three exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than two exogenous nucleic acids that encode a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than one exogenous nucleic acid that encodes a lipid pathway enzyme. In some embodiments, the bacterial cell comprises no more than eight exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than seven exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than six exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than four exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than three exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than two exogenous nucleic acids that encode a protein. In some embodiments, the bacterial cell comprises no more than one exogenous nucleic acid that encodes a protein.
In some embodiments, the molecule is chosen from one or more alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or dicarboxylic acid (diacid). In some embodiments, the method produces a lipid or mixture of lipids at a quantity higher than the quantity of lipid or mixture of lipids in the same bacterial cell population not comprising the exogenous nucleic acids described herein. In some embodiments the one or more lipids comprise a quantity of at least one alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid at a level higher than the quantity of the alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid in the same microorganism not comprising the exogenous nucleic acid sequences. In some embodiments, the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 10% of the total percentage of cellular dry matter. In some embodiment, the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 20% of the total percentage of cellular dry matter. In some embodiment, the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 30% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 40% of the total percentage of cellular dry matter. In some embodiment, the method comprises a population of microorganisms or bacterial cell described herein that produces and/or secretes lipids of a weight equal to or greater than 50% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cells described herein that produces and/or secretes lipids of a weight equal to or greater than 60% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cells described herein that produces and/or secretes lipids of a weight equal to or greater than 70% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 75% of the total percentage of cellular dry matter. In some embodiment, the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 80% of the total percentage of cellular dry matter. In some embodiments, the method comprises a population of microorganisms or bacterial cell described herein that produces of secretes lipids of a weight equal to or greater than 85% of the total percentage of cellular dry matter. In some embodiments, the bacterial cell or composition comprising the bacterial cell produces and/or secretes at least 10% of the total percentage of the cellular dry matter or 10% by weight. In some embodiment, the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 5% of the lipids have carbon backbones from 8 to 18 carbon atoms in length. In some embodiment, the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 10% of the lipids have carbon backbones from 8 to 18 carbon atoms in length. In some embodiments, the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 15% of the lipids have carbon backbones from 8 to 18 carbon atoms in length. In some embodiments, the method comprises a population of microorganisms comprising a bacterial cell described herein that produces or secretes lipids, wherein at least 20% of the lipids have carbon backbones from 8 to 18 carbon atoms in length.
In some embodiments of the invention, the invention relates to a method of fixing carbon from a gaseous feedstock containing carbonaceous molecules, wherein the method comprises the step of exposing a composition comprising a bacterial cell to syngas and/or gaseous CO2 and/or gaseous H2; wherein the bacterial cell comprises at least one exogenous nucleic acid sequence. In some embodiments the exogenous nucleic acid sequences are fatty acid acyl-ACP reductase or a fatty acid aldehyde decarbonylase. In some embodiments of the method, the bacterial cell comprises at least a first and a second exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase. In some embodiments, the bacterial cell is Rhodococcus opacus or the population of microorganisms comprises a Rhodococcus cell. In some embodiments, the bacterial cell is Cupriavidus necator or the population of microorganisms comprises a Cupriavidus cell. In some embodiments, the bacterial cell comprises at least a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acid sequences that encode a lipid pathway enzyme. In some embodiments the bacterial cell comprises at least a first and a second exogenous nucleic acid sequence but no more than five exogenous nucleic acid sequences, wherein the first exogenous nucleic acid sequence encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.
In some embodiments, the invention relates to a method of producing one or more alkene, alkyne, unsaturated fatty acid, hydroxyacid and/or diacid or any combination thereof comprising exposing a bacterial cell to syngas and/or gaseous CO2 or a mixture of gaseous CO2 and gaseous H2; wherein the bacterial cell is capable of fixing gaseous CO2 into one or more alkene, alkyne, unsaturated fatty acid, hydroxy acid and/or diacid and wherein the microorganism comprises at least a first exogenous nucleic acid and a second exogenous nucleic acid, wherein the first exogenous nucleic acid encodes fatty acid acyl-ACP reductase and the second exogenous nucleic acid encodes fatty acid aldehyde decarbonylase. In some embodiments, the first and second exogenous nucleic acids are heterologous nucleic acid sequences. In some embodiments, the bacterial cell comprises at least a first, a second, and a third exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a fatty acid acyl-ACP reductase, the second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and the third exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the bacterial cell comprises at least a first exogenous nucleic acid sequence, wherein the first exogenous nucleic acid sequence encodes a thioesterase. In some embodiments, the bacterial cell comprises no more than five exogenous nucleic acid sequences that encode a lipid pathway enzyme.
In some embodiments, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes an increase in the ratio of C12:C14:C16 lipids or hydrocarbons produced or secreted by the cell as compared to the C12:C14:C16 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of C12:C14:C16 lipids or hydrocarbons produced or secreted by the cell as compared to the C12:C14:C16 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In some embodiments, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes an increase in the ratio of C13:C15:C17 lipids or hydrocarbons produced or secreted by the cell as compared to the C13:C15:C17 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of C13:C15:C17 lipids or hydrocarbons produced or secreted by the cell as compared to the C13:C15:C17 lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In some embodiments, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes an increase in the ratio of odd-carbon numbered lipids or hydrocarbons produced or secreted by the cell as compared to the odd-carbon numbered lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In some embodiments, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of odd-carbon numbered lipids or hydrocarbons produced or secreted by the cell as compared to the odd-carbon numbered lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the lipid pathway enzyme. In one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a decrease in the ratio of even:odd carbon numbered lipids or hydrocarbons produced or secreted by the cell as compared to the ratio of even:odd carbon numbered lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the one or more lipid pathway enzymes. In one embodiment, the one or more exogenous nucleic acid sequence encodes at least one lipid pathway enzyme that causes a increase in the ratio of even:odd carbon numbered lipids or hydrocarbons produced or secreted by the cell as compared to the ratio of even:odd carbon numbered lipids or hydrocarbons produced or secreted by the same cell not transformed with the nucleic acid sequence that encodes the one or more lipid pathway enzymes.
In some embodiments the exogenous gene or genes codes for enzymes or proteins including but not limited to one or more of the following: an acyl carrier protein, a TE, a FAR, a FadR, a FAD, a fatty aldehyde reductase, a cytochrome P450 enzyme, a NADH or NADPH cytochrome P450 reductase, a desaturase, a hydroxylase, and an antibiotic resistance enabling protein. In some embodiments, the coding sequence comprises an exogenous nucleic acid sequence that encodes a TE that catalyzes hydrolysis of one or more fatty acyl-ACP substrates with chain lengths ranging over C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18. In some embodiments, the cell comprises a plasmid that contains one or more exogenous nucleic acid sequences that encode an amino acid sequence for an enzyme or protein such as but not limited to one or more of the following: an acyl carrier protein, a TE, a FAR, a FadR, a FAD, a fatty aldehyde reductase, a cytochrome P450 enzyme, a NADH or NADPH cytochrome P450 reductase, a desaturase, a hydroxylase, and an antibiotic resistance enabling protein. In some embodiments, the one or more exogenous nucleic acid sequences comprise SEQ ID NO:5 or a functional fragment thereof that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO:5. In some embodiments, the one or more exogenous nucleic acid sequences comprise SEQ ID NO:6 or a functional fragment thereof that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to SEQ ID NO:6.
In further embodiments, at least one coding sequence of the at least one exogenous nucleic acid sequence encodes a lipid pathway enzyme. In some embodiments, the at least one coding sequence of the at least one exogenous nucleic acid sequence encodes a lipid modification enzyme. In some embodiments, the composition or cell comprises a nucleic acid that encodes at least one fatty acid decarbonylase, at least one fatty acid reductase, a thioesterase, or any combination of any one more full-length lipid pathway enzymes or functional fragments thereof. In some embodiments the one or more exogenous nucleic acid sequences are integrated into the genome of the cell. In some embodiments, the one or more exogenous nucleic acid sequences are on an episomal plasmid within the transformed host cell.
Methods of Isolation and Purification
Following the methods of the present invention microorganisms are grown and maintained for the production of lipids in a medium containing a gaseous carbon source, such as but not limited to syngas or producer gas, in the absence of light; such growth is known as chemotrophic growth. In some embodiments, the invention relates to methods of cultivating oleaginous cells for the large-scale production of oil and/or fuel. In some embodiments, the invention relates to methods of cultivating oleaginous cells in bioreactors 50,000 liters or greater in volume, which are conventionally constructed out of low cost, sturdy, and opaque materials such as steel or reinforced concrete or earthworks. The size, depth, and construction of such bioreactors dictate that the cells will be grown in near or total darkness. In some embodiments, the oleaginous microorganisms are cultured for the synthesis of lipids in accordance with the methods of the present invention in a medium containing gaseous inorganic carbon, such as but not limited to syngas or producer gas, as the primary or sole carbon source, and without any exposure to light. This type of growth is known as chemoautotrophic growth.
To give an illustration, a bioreactor containing nutrient medium is inoculated with of oleaginous bacterial cells; generally there will follow a lag phase prior to the cells beginning to double. After the lag phase, the cell doubling time decreases and the culture goes into the logarithmic phase. The logarithmic phase is eventually followed by an increase of the doubling time that, while not intending to be limited by theory, is thought to result from either a depletion of nutrients including nitrogen sources, or a rise in the concentration of inhibitory chemicals, or quorum sensing by the microbes. The growth slows down and then ceases when the culture goes into the stationary phase. In order to harvest cell mass with high lipid content, the culture is generally harvested late in the logarithmic phase or in the stationary phase. In some embodiments, the cells are harvested in logarithmic phase. In some embodiments, the cells are harvested in stationary phase. The accumulation of lipid can generally be triggered by the depletion of the nitrogen source or another key nutrient excepting the carbon or the energy source (e.g. hydrogen). This signals the cells to store lipids produced from the excess carbon and energy sources. Optimization of lipid production and the targeting of specific lipid distributions can be achieved by control of bioreactor conditions and/or nutrient levels and/or through genetic modifications of the cells. In some embodiments the lipid production and distribution of lipid molecules produced is optimized through one or more of the following: control of bioreactor conditions, control of nutrient levels, genetic modifications of the cells.
The synthesis of lipids by the microbes disclosed in the present invention can happen during the logarithmic phase and afterwards during the stationary phase when cell doubling has stopped provided there is an ample supply of carbon and energy sources,
In some embodiments, microorganisms grown using conditions described herein and known in the art comprise at least 20% lipid content by weight, but under chemotrophic conditions, comprise at least 10% lipid content by weight. In some embodiments, under chemotrophic conditions, the microorganisms of the present invention comprise at least about 10, 15, 20, 25, 30, 35, or 40% by weight of lipids, at least about 50% by weight, or at least about 60% by weight of lipids. Improved lipid yield and/or lower production costs can be achieved by controlling process parameters. In certain embodiments, a bacterium is grown in a nutrient media and/or gas mix having a nitrogen, oxygen, phosphorous, or sulfur limitation, while a gaseous carbon and energy source such as syngas is provided in excess. Lipid yield is generally higher in microbial cultures grown with a nitrogen limitation versus microbial cultures grown without nitrogen limitation. In certain embodiments, lipid yield rises by at least: 10%, 50%, 100%, 200%, 500%, or 1000%. The microbial growth can occur with nutrient limitation for a part or for all of the fermentation run. Feeding an excess of energy and carbon source to a population of oleaginous microbes, but little or no nitrogen, can produce a rise in cellular lipid content. In some embodiments, microbial growth occurs on limited amounts of nitrogen or in the complete absence of nitrogen.
Genes are well known in the art that code for cofactors useful in the present invention, or that are involved in synthesizing such cofactors.
In another embodiment, genes that code for cofactors useful in the present invention, or that are involved in synthesizing such cofactors, are put in oleaginous bacteria, using the constructs and methods such as described above. Lipid yield is improved in another embodiment by growing an oleaginous bacteria with one or more lipid pathway enzyme cofactor(s) added to the culture environment. The lipid yield is generally improved in the presence of a certain concentration of the cofactor(s) compared to lipid yield without supplemental cofactor(s). In some embodiments, the cofactor(s) are delivered to the culture by having a microbe (e.g., bacteria) present in the culture that contains an exogenous gene coding for the cofactor(s) at a concentration sufficient to increase lipid yield as compared to the lipid yield of the microbe in the absence of the cofactor. Cofactor(s) may also be delivered to a culture by having a microbe (e.g., bacteria) present in the culture that contains an exogenous gene that coding for a protein involved in the cofactor synthesis. In some embodiments, any vitamin needed for the proper function of a lipid pathway enzyme including biotin and/or pantothenate is included in the culture environment.
The specific examples of bioreactors, culture conditions, heterotrophic and chemotrophic growth, maintenance, and lipid production methods described herein can be combined in any suitable manner to improve efficiencies of microbial growth and lipid and/or protein production.
In another aspect of the invention, the invention relates to a method of producing a molecule or mixture of molecules in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In another aspect of the invention, the invention relates to a method of producing a hydrocarbon or mixture of hydrocarbons in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In another aspect of the invention, the invention relates to a method of producing a lipid or mixture of lipids in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In another aspect of the invention, the invention relates to a method of producing an alkene or mixture of alkenes in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In another aspect of the invention, the invention relates to a method of producing an alkyne or mixture of alkynes in a microorganism population comprising the cell or the composition described herein, wherein the method comprises: culturing a population of microorganisms comprising the cell or the composition described herein in a feedstock comprising syngas and/or gaseous CO2 and/or a mixture of CO2 gas and H2 gas.
In some embodiments, the methods of the claimed invention do not rely on desulfonation to produce and/or secrete one or more hydrocarbons. In some embodiments, an exogenous nucleic acid is introduced into the cells of the claimed invention to silence or disrupt transcription of endogenous genes of the cell that encode enzymes capable of desulfonation of commercial surfactants under conditions and for a time period sufficient for growth of the cell with a gaseous feedstock comprising a gas comprising carbon.
In some embodiments, the feedstock does not include linoleic acid.
The following documents are incorporated herein by reference in their entirety for all purposes: U.S. Provisional Patent Application No. 61/328,184, filed Apr. 27, 2010 and entitled “USE OF OXYHYDROGEN MICROORGANISMS FOR NON-PHOTOSYNTHETIC CARBON CAPTURE AND CONVERSION OF INORGANIC CARBON SOURCES INTO USEFUL ORGANIC COMPOUNDS”; International Patent Application Serial No. PCT/US2010/001402, filed May 12, 2010, entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGNISMS FOR THE CHEMOSYTHETIC FIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTO ORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS”; and U.S. Patent Application Publication No. 2010/0120104, filed Nov. 6, 2009, entitled “BIOLOGICAL AND CHEMICAL PROCESS UTILIZING CHEMOAUTOTROPHIC MICROORGNISMS FOR THE CHEMOSYTHETIC FIXATION OF CARBON DIOXIDE AND/OR OTHER INORGANIC CARBON SOURCES INTO ORGANIC COMPOUNDS, AND THE GENERATION OF ADDITIONAL USEFUL PRODUCTS.
Doan T T P, Carlsson A S, Hamberg M, Bulow L, Stymne S, Olsson P, Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli, J Plant Phys 166 (2008):787-96.
Kavanagh K L, Jornvall H, Persson B, Oppermann U, The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes, Cell Mol Life Sci 65 (2008) 3895-3906.
Labesse G, Vidal-Cros A, Chomilier J, Gaudry M, Mornon J-P, Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the ‘RED’ family), Biochem J (1994) 304:95-99.
The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention. Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
EXAMPLE 1
Characterization of Organisms Sharing High 16SrRNA Sequence Similarity
To identify organisms closely related to R. opacus strain (DSM43205), a basic local alignment search (BLASTR) with the BLASTN programs search of nucleotide databases using the 16S rRNA (NR_026186.1) was carried out. The phylogenetic relationships, based on the 16S rRNA gene sequence homology, between the tested strain and the reference strains of the suborder corynebacterineae (corynebacterium, gordoniaceae, mycobacteriaceae and nocardiaceae) and the family burkholderiaceae (genus cupriavidus and ralstonia) are shown in FIG. 2. The nocardiaceae are related and form two clusters of organisms: clusturel that contains 20 organisms from the genus nocardia and rhodococcus and cluster 2 that contains 3 R. opacus strains (DSM43205, GM14 and DSM43206). The gordoniaceae, mycobacteriaceae and burkholderiaceae form 3 separated groups (1, 2 and 3). The gram positive chemoautotroph lipid accumulating strain R. opacus (DSM43205; NR_026186.1) exhibits high sequence similarity to cluster 1 (94.3-99.1%) and to the gram positive groups 1 and 2 (92.7-93.5% and 93.3-93.6% respectively) (FIGS. 3 and 4). The sequence similarity to the gram-negative chemoautotroph poly(3-hydroxybutyrate) (PHB) accumulating strains in group 3 is 73.7%.
EXAMPLE 2
Plasmid Design and Construction
To generate an E. coli Rhodococci shuttle vector suitable for electroporation, the plasmid pSeqCO1 (SEQ ID: 01) was constructed with the genetic elements described in FIG. 10A. pSeqCO1 consists of the replication gene operon, ampicillin and kanamycin resistance genes, LacZ operon and the multiple cloning site as described in FIG. 10B and FIG. 11A. For replication in Rhodococci, the DNA fragment of the repAB operon (1744 bp downsteam from the XhoI restriction site in the native pKNR01 plasmid of the bacteria Rhodococcus opacus B4; Na et al. 2005, J Biosci Bioeng. 99: 408-414) was synthesized with the restriction sites KpnI and SalI and cloned into PUC 18 digested with KpnI and SalI. The resultant vector was digested with SpeI and BglII and ligated with the PCR product of the Kanamycin resistance gene from pBBR1MCS-2 (Kovach et al. 1995 Gene 166: 175-176) digested with the engineered restriction sites SpeI and BglII to give pSeqCO1.
To generate an E. coli-cupriavidus shuttle vector suitable for electroporation and bacterial conjugation, the plasmid pSeqCO2 (SEQ ID: 02) was used with the genetic elements described in FIG. 10A. pSeqCO2 (SEQ ID: 02; FIGS. 10 and 11B) is the plasmid pBBR1MCS-2 described in Kovach et al. (1995 Gene 166: 175-176) that contains the IncQ like replication gene, Mob gene that mobilized when the RK2 transfer functions are provided in trans, kanamycin resistance gene, LacZ operon and the multiple cloning site as described in FIG. 10B and FIG. 11B.
Pver1 (SEQ ID: 03; FIGS. 10 and 11C) is an E. coli-cupriavidus-Rhodococci shuttle vector suitable for electroporation and bacterial conjugation. The plasmid was generated by cloning the repAB operon (described in pSeqCO1) into pSeqCO2 using the KpnI and SalI restriction sites.
Pver2 (SEQ ID: 04; FIGS. 10 and 11D) is an E. coli-cupriavidus-Rhodococci shuttle vector suitable for electroporation and bacterial conjugation. The plasmid was generated by cloning the synthesized chloramphenicol gene (Alton and Vapnek Nature 1979 282: 864-869) with the engineered restriction sites SalI and HindIII into Pver1.
The genes FadDR (SEQ ID: 05) and Fad (SEQ ID: 06) and the rbcLXS promoter (SEQ ID: 7) were PCR amplified from the cyanobacterium Synechocystis sp. PCC 6803 genome and cloned into gateway plasmid to give pFUEL. A 4 kBp XhoI BamHI fragment that contains FadDR, Fad and rbcLXS was rescued from pFUEL and cloned into pSeqCO2 digested XhoI BamHI with to give pSeqCO2::FUEL (FIG. 14).
EXAMPLE 3
Microorganism Transformation
Transformation of Rhodococci was carried out using the plasmids pSeqCO1 and pVer1 (FIG. 12) as described below.
Rhodococci competent cells were prepared by incubating a single colony 2 ml NB medium (5 g/L peptone, 1 g/L meat extract, 2 g/L yeast extract, 5 g/L NaCl; pH=7.0±0.2) at 30° C. overnight. One ml was inoculated to 50 ml NB medium supplemented with 0.85% (w/v) glycine and 1% (w/v) sucrose in a 250 ml baffled Erlenmeyer Flask and incubated to a cell density of O.D600=0.5. Cells were collected by centrifugation at 3,000×g for 10 min at 4° C. and washed 3 times with 50 ml (each) of sterile ice-cold double distilled water (ddH2O). Cells were concentrated 20-fold by re-suspending the collected cells in 2.5 ml of ddH2O and 400 μl aliquots stored in1.5 ml tube at −70° C. Electroporation was carried out by thawing the competent cells on ice and mixing with the plasmid DNA (final concentration 0.1-0.25 μg/ml). The competent cells and plasmid DNA mixture was incubated at 40° C. for 5 min, transferred into 0.2 cm width and electroporated using a single-pulse electroporation (10 kV/cm, 600Ω, 25 μF and 3-5 ms pulse time). The pulsed cells were regenerated at 30° C. for 4 h (DSM 44193) and 6 h (DSM 43205) in the presence of 600 μl NB. Transformants were selected after cultivation for 3-4 days at 30° C. on NB-agar plate containing kanamycin (75 μg/ml). As shown in FIG. 12, the plasmids pSeqCO1 and pVer1 confer resistance to kanamycin (75 μg/ml) in transformed R. opacus strains (44193 and 43205). Untransformed R. opacus strains (44193 and 43205) (NC) were sensitive to the concentration described above.
Transformation of genus cupriavidus was carried out using the plasmids pSeqCO2 (FIG. 12) as described below.
Cupriavidus necator (DSM531) competent cells were prepared by incubating a single colony in 5 ml NR medium (10 g/l polypeptone, 10 g/l yeast extract, 5 g/l beef extract and 5 g/l ammonium sulfate; pH 7.0) at 30° C. overnight. The pre-culture was inoculated into 100 ml of fresh NR medium and incubated to a cell density of O.D600=0.8. Cells were collected by centrifugation at 3,000×g for 10 min at 4° C. and washed 3 times with 50 ml (each) of sterile ice-cold ddH2O. The collected cells were re-suspended in 400 μl of 10% (v/v) sterile glycerol in sterile ice-cold ddH2O and stored in 50 μl aliquots at −70° C.
For electroporation, the competent cells were thawed on ice, transferred into 0.2 cm width of ice cold cuvette and gently mixed with 1 μg of plasmid DNA. Cells were electroporated using a single-pulse electroporation (11.5 kV/cm, 25 μF and 5 ms pulse time). The pulsed cells were transferred into 1 ml of fresh NR medium and culture for 2 h at 30° C. Transformants were selected after cultivation for 48 h at 30° C. on NR-agar plate containing kanamycin (200 μg/ml). As shown in FIG. 12, the plasmid pSeqCO2 confers resistance to kanamycin (200 μg/ml) in transformed Cupriavidus necator (DSM531). Untransformed Cupriavidus necator (DSM531) cells (NC) were sensitive to the concentration described above.
EXAMPLE 4
Inoculation and Growth Conditions
Organisms from the genus rhodococcus and from the genus cupriavidus were tested for their ability to grow on different carbon sources (FIG. 5). Colonies from strains grown on LB agar plates at 30° C. were transferred into flasks containing 10% (v/v) of the indicated media for 3-20 days at 30° C. and 250 rpm. R. opacus strain DSM 44193 exhibited growth only under heterotrophic growth conditions as measured by optical density (OD) at 650 nm on MSM medium (1 L Medium A: 9 g Na2HPO412H2O, 1.5 g H2PO4, 1.0 g NH4Cl and 0.2 g MgSO4.7H2O per 1 L; 10 ml Medium B: 50 mg Ferric ammonium citrate and 100 mg CaCl2 per 100 ml; 10 ml Medium C: 5 g NaHCO3 per 100 ml; and 1 ml Trace Mineral Solution: 100 mg ZnSO4.7H2O, 30 mg MnCl2. 4H20, 300 mg H3BO3, 200 mg COCL2.6H20, 10 mg CuCl2.2H2O, 20 mg NiCl2.6H2O and 30 mg Na2MoO4.2H2O per 1 L) supplemented with 40 g/L glucose. R. opacus strain DSM 43205 showed identical growth rates under heterotrophic conditions reaching O.D=9.0. Strain DSM 43205 was also able to grow on chemoautotrophic conditions (MSM medium supplemented with 66.7% H2, 9.5% CO2, 5% O2 and 18.8% N2) and heterotrophically on a single carbon compound as the solely carbon source (MSM medium supplemented with 25 g/1 methanol). Rhodococcus sp. (DSM 3346) exhibited growth under heterotrophic conditions and chemoautotrophic conditions (DSMZ Medium 81: 1 L of Mineral Medium for chemolithotrophic growth: 2.9 g Na2HPO4.2H2O, 2.3 g KH2PO4, 1.0 g NH4Cl, 0.5 g MgSO4.7H2O, 0.5 g NaHCO3, 0.01 g CaCl.2H2O and 0.05 g Fe(NH4) citrate per 1 L; and 5 ml Trace Mineral Solution, supplemented with 80% H2, 10% CO2 and 10% O2). Cupriavidus necator (DSM 531) was able to grow under heterotrophic and chemoautotrophic conditions (media described for Strain DSM 43205) (FIG. 5 and FIG. 22). Cupriavidus necator (DSM 531) transformed with pSeqCO2 was able to grow on LB media supplemented with 300 400 and 500 μg/mlkanamycin exhibiting O.D600 of 1.47, 1.52 and 1.51 respectively (FIG. 13). Untransformed cells exhibited growth on control (LB only) and some growth on 300 μg/mlkanamycin while no growth was detected on 400 and 500 μg/mlkanamycin.
EXAMPLE 5
Lipid Profiles
Production of Fatty Acid
Under heterotrophic growth conditions strains DSM 44193, DSM 43205, DSM 3346 and DSM 531 produce lipid (FIG. 6). Lipid content determined by gas chromatography analysis of cells harvested after 72 hr (unless otherwise indicated) showed over 19% of cellular dry matter (CDM) determined gravimetrically for strains DSM 44193, DSM 43205 and DSM 3346. The lipid content of DSM 43205 was higher than 10% of under chemoautotrophic conditions. Under heterotrophic growth conditions DSM 44193 produces 32%, 26% and 21% of 16, 17 and 18-carbon fatty acid respectively (FIG. 7). DSM43205 produces similar amounts of 16, 17 and 18-carbon fatty acid (30%, 24% and 32% respectively) (FIG. 8A). Chemoautotrophic growth condition significantly reduces the 17-carbon fatty acid abundance (6%) and maintains similar levels of 16 and 18-carbon fatty acid (36% and 27% respectively) (FIG. 8B). DSM3346 exhibits similar fatty acid distribution of 16, 17 and 18-carbon fatty acid (39%, 24% and 25% respectively) (FIG. 9A) under heterotrophic growth. Chemoautotrophic growth condition significantly increases the 16-carbon fatty acid levels (66%) and reduces the 17 and 18-carbon fatty acid levels (4%, 14%)(FIG. 9B).
EXAMPLE 6
Production of Hydrocarbons
To redirect carbon flux from fatty acid toward hydrocarbon biosynthesis, the genes Fatty acyl-CoA/Fatty acyl-ACP reductase (FadR) and Fatty aldehyde decarbonylase (FAD) from the decarbonylation pathway of cyanobacteria (indicated in red) were expressed in Cupriavidus necator (DSM 531).
The plasmid pSeqCO2::FUEL (FIG. 14) described in the text was introduced into Cupriavidus necator (DSM 531) as described above and 2 independent transformants (Cn-FUEL2.1 and Cn-FUEL2.2) were selected. One hundred ml of Cn-FUEL2.1, Cn-FUEL2.2 and control cells (empty plasmid: Cn-P) were incubated on LB medium with 400 μg/mlkanamycin for 30 hr. Cells were harvested at 3,000×g for 10 min at 4° C. and pellet was analyzed by GC/MS. Cn-FUEL2.1 (FIG. 15A) and Cn-FUEL2.2 showed a specific peak at 45.00 min compared to control Cn-P (FIG. 15B) indicating the presence of hydrocarbons in the engineered strains. Cn-FUEL2.1, Cn-FUEL2.2 produced high levels (over 2%) of unique molecules such as: Spiro[4.5]decane, Bicyclo[10.8.0]eicosane, cis,cis-1,6-Dimethylspiro[4.5]decane, 1,19-Eicosadiene, Cyclooctacosane, Bicyclo[10.8.0]eicosane, 1-Pentadecyne, 1-Pentadecyne, Heptacosyl acetate, 5-Cyclohexyl-1-pentene, 1-Hexadecyne and Cyclodecacyclotetradecene, -eicosahydro (FIGS. 16 and 17).
The effect of the production of hydrocarbons on fatty acid distribution is shown in FIG. 18. The fatty acids profile of 2 independent control experiments (Cn-P) shows predominantly 16-carbon (63% and 61%) and 18-carbon (33% and 32%) fatty acids. In contrast, Cn-FUEL2.1 and Cn-FUEL2.2 exhibit significantly lower levels of 16-carbon (29%, 33% respectively) and 18-carbon (3% and 2% respectively) fatty acids. Cn-FUEL2.1 and Cn-FUEL2.2 show a significant increase in the 15-carbon fatty acid (50% and 45% respectively) compared to 0.08% and 0.09% in the control strains Cn-P.
EXAMPLE 7
Purification
Purification Hydrocarbons
To produce hydrocarbons in bacteria, genes from the decarbonylation pathway of cyanobacteria, including but not limited to, the FadR (SEQ ID: 05) and FAD (SEQ ID: 06) genes will be cloned into pVer2 (SEQ ID: 04) to give pVer2::FUEL. Bacteria, including but not limited to, R. opacus strain (DSM43205) will be transformed with the plasmid pVer2::FUEL by electroporation and grown in 100 ml LB medium supplemented with 75 μg/mlkanamycin for 30 hr. The cells (2×50 ml) will be harvested at 3,000×g for 10 min at 4° C. and the pellet and the supernatant further analyzed. Analysis of hydrocarbons from the cell pellet will be carried out in 25 mm×150 mm glass tube in the presence of 50 μL of Eicosane standard (approx 200 μg/ml) and 50 μl lipid standard ( ̃200 ug/ml). Pellet will be extracted with 5 mL chloroform, 10 ml methanol, 4 ml phosphate buffer (phosphate buffer reagent: 50 mM, pH 7.4, 8.7 g K2HPO4 in 1 L water, and about 2.5 ml 6N HCl to adjust pH=7.4, and 50 ml chloroform per 1 L buffer). The mixture will be vortexed for 30 sec, sonicated for 2 min and incubated in dark for at least 3 hr. Phases will be separated in the presence of 5 mL chloroform and 5 ml ddH2O, vortexed and spun down 2000 rpm for 1 min. The bottom layer will be transferred with a glass Pasteur pipette to clean 16 mm×125 mm glass tube with Teflon-lined screw top and dried under N2. The dried extract will be re-suspended in hexane and analyzed by Gas Chromatography for the presence of hydrocarbons, including but not limited to 1-Hexadecyne.
Purification of Fatty Acids
To modify the fatty acid distribution in bacteria, thioesterases that regulate the fatty acid chain length, including but not limited to the YP_002784058.1 gene will be cloned into pVer2 (SEQ ID: 04) to give pVer2::TE. Bacteria, including but not limited to, R. opacus strain (DSM43205) will be transformed with the plasmid pVer2::TE by electroporation and grown in 100 ml LB medium supplemented with 75 μg/mlkanamycin for 30 hr. The cells (2×50 ml) will be harvested at 3,000×g for 10 min at 4° C. and the pellet and the supernatant further analyzed. Analysis of fatty acids from the cell pellet will be carried out in 25 mm×150 mm glass tube in the presence of 504 of Eicosane standard (approx 200 μg/mL) and 50 μL lipid standard ( ̃200 ug/ml). Pellet will be extracted with 5 ml chloroform, 10 ml methanol, 4 ml phosphate buffer (phosphate buffer reagent: 50 mM, pH 7.4, 8.7 g K2HPO4 in 1 L water, and about 2.5 mL 6N HCl to adjust pH=7.4, and 50 ml chloroform per 1 L buffer). The mixture will be vortexed for 30 sec, sonicated for 2 min and incubated in dark for at least 3 hr. Phases will be separated in the presence of 5 ml chloroform and 5 ml ddH2O, vortexed and spun down 2000 rpm for 1 min. The bottom layer will be transferred with a glass Pasteur pipette to clean 16 mm×125 mm glass tube with Teflon-lined screw top and dried under N2. The dried extract will be re-suspended 1.5 ml of a 10:1:1 mixture of Methanol:CHCl3:concentrated HCl, vortexed and incubated in 60° C. for 14-16 hr (overnight). The extracts will be cooled and 2 ml of ddH2O and 2 ml of hexane will be added, vortexed and centrifuged for 5 min at 2000 rpm for phase separation. The top hexane layer will be transferred to clean 16 mm tube. An additional two hexane extractions (vortex, centrifugation and phase separation) will be carried out in the extract tube. The hexane extracts will be dried in a GC vial and analyzed by Gas Chromatography for the presence of fatty acids, including but not limited to dodecanoic acid.
EXAMPLE 8
Dicarboxylic acids with targeted chain length. Genetically engineer bacteria from the suborder corynebacterineae or the family burkholderiaceae to express thioesterases which yield different length fatty acids. For example the YP_002784058.1 gene mentioned previously or:
















C


UniProt Entry
Protein name
Organism
length







FATB_GOSHI
Myristoyl-acyl carrier protein
Gossypium
16:0



thioesterase
hirsutum



FATB_UMBCA
Lauroyl-acyl carrier protein
Umbelliularia
12:0



thioesterase
californica



FATB_CINCA
Myristoyl-acyl carrier protein
Cinnamomum
14:0



thioesterase
camphora



FATA_CORSA
Oleoyl- acyl carrier protein
Coriandrum
18:0



thioesterase
sativum



FATB_CUPHO
Myristyl- acyl carrier protein
Cyphea
16:0



thioesterase
hookeriana









Thioesterases generating shorter chain fatty acids (e.g., C10:0 or C12:0) will be identified and incorporated into the bacteria from the suborder corynebacterineae and the family burkholderiaceae.
Extract the resulting lipids and provide as the sole source of carbon to a culture of Candida tropicalis ATCC 20336, which contains the relevant enzymatic pathways to produce the alpha, omega-dicarboxylic acids. Identify and purify dicarboxylic acid end products from second culture.
EXAMPLE 8
Dicarboxylic Acids with Targeted Chain Length
As in previous example, engineer host strain bacteria from the suborder corynebacterineae or the family burkholderiaceae to express thioesterases which yield targeted chain length fatty acids.
Also, engineer into host strain cytochrome P450 pathway from Candida tropicalis, including the CYP52A genes with NADPH cytochrome P450 reductase to generate dicarboxylic acid from the fatty acids. Craft et al. have identified the key genes for generation of alpha, omega-dicarboxylic acids in Candida tropicalis: CYP52A13, CYP52A14, CYP52A17, CYP52A18, and CYP52A12 along with the corresponding reductase (Craft 2003).
Perform a single culture, which generates appropriate length fatty acids, then modifies to attach a second carboxylic acid.
EXAMPLE 9
Dicarboxylic Acids
Culture the hyperthermophilic archaeon Pyrococcus furiosus in order to generate the dicarboxylic acids described in Carballeira et al. (Carballeira 1997). Determine genetic machinery for generating these dicarboxylic acids, and compare P furiosus genome with the bacteria from the suborder corynebacterineae and the family burkholderiaceae genomes. Move the relevant genetic modules from the P furiosus into the bacteria from the suborder corynebacterineae and the family burkholderiaceae in order to post-process lipids into dicarboxylic acids. This can be combined with genes which produce shorter fatty acids through the appropriate thioesterases.
EXAMPLE 10
Hydroxy-acids. For generating omega-hydroxylated fatty acids, incorporate vicia sativa P450-dependent fatty acid omega hydroxylase into the bacteria from the suborder corynebacterineae and the family burkholderiaceae cell line. This enzyme hydroxylates myristic acid (C14), lauric acid (C12), pamitic acid (C16), but not oleic acid (C18).
EXAMPLE 11
Hydroxy-acids. For generating in-chain hydroxylated fatty acids, incorporate CYP81B1 (H tuberosus) or CYP709C1 (unknown) fatty acid hydroxylases into the bacteria from the suborder corynebacterineae and the family burkholderiaceae cell line. The CYP81B1 enzyme omega-1 and omega-5 mono-hydroxylates capric (C10:0), lauric (C12:0), and myristic (C14:0) (Pompon 1996). The CYP709C1 gene hydroxylates the omega-1 and omega-2 positions independent of chain length (Kandel 2005).
Specific preferred embodiments of the present invention have been described here in sufficient detail to enable those skilled in the art to practice the full scope of invention. However it is to be understood that many possible variations of the present invention, which have not been specifically described, still fall within the scope of the present invention and the appended claims. Hence these descriptions given herein are added only by way of example and are not intended to limit, in any way, the scope of this invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.

## Claims

1. A composition comprising a microorganism in a culture medium that is in contact with a gaseous feedstock that comprises:
CO2; H2 and/or CO; and O2,
wherein said culture medium does not comprise an organic carbon source as a primary source of carbon for microbial growth or biosynthesis,
wherein said primary or sole source of carbon for microbial growth or biosynthesis is inorganic carbon,
wherein said microorganism is a member of the genus Cupriavidus and is capable of chemoautotrophic metabolism;
wherein said microorganism chemoautotrophically incorporates CO2, from said gaseous feedstock into organic compounds comprising one or more lipids or hydrocarbons in said culture medium,
wherein said H2 and/or CO serves as an inorganic electron donor and is utilized by said microorganism as an energy source and as a reducing agent for said production of said organic compounds,
wherein said O2 serves as an electron acceptor for generation of intracellular energy carriers,
wherein H2O is produced in the generation of intracellular energy carriers in said microorganism via an oxyhydrogen reaction,
wherein less than 10% by weight of said organic compounds produced is acetic acid or butyric acid and wherein less than 10% by weight of said hydrocarbons produced is methane,
wherein less than 10% by weight of said organic compounds produced are organic acids with carbon chain length of four carbons or less, and
wherein said microorganism comprises at least a first exogenous nucleic acid sequence encoding a fatty acid aldehyde acyl-ACP reductase and a fatty acid aldehyde decarbonylase, or comprises at least a first exogenous nucleic acid encoding a thioesterase, or comprises at least a first exogenous nucleic acid encoding a fatty acyl-CoA/fatty acyl-ACP reductase, and
wherein said one or more lipids or hydrocarbons comprises one or more of Spiro[4.5]decane; 11-Hexacosyne; 9-Tricosene, (Z)-; Triacontyl acetate; 1-Heptacosanol; 5-Nonadecen-1-ol; Nonadecyl trifluoroacetate; Bicyclo[10.8.0]eicosane; (E)-, cis,cis-1,6-Dimethylspiro[4.5]decane; 1,19-Eicosadiene; Cyclododecene, 1-methyl-; Cyclooctacosane; Bicyclo[10.8.0]eicosane, cis-; 1-Pentadecyne; Heptacosyl acetate; Cyclotetracosane; 5-Cyclohexyl-1-pentene; Cyclododecene, 1-methyl-; 1-Hexadecyne; 1,21-Docosadiene; Cyclodecacyclotetradecene, -eicosahydro-; 17-Pentatriacontene; and Squalene.

2. The composition of claim 1, wherein said microorganism comprises at least a first and a second exogenous nucleic acid sequence but no more than five exogenous nucleic acid sequences.

3. The composition of claim 1, wherein said exogenous nucleic acid sequence consists of a first, a second, and a third exogenous nucleic acid sequence, wherein said first exogenous nucleic acid sequence encodes fatty acid aldehyde acyl-ACP reductase, said second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase, and said third exogenous nucleic acid sequence encodes a thioesterase.

4. The composition of claim 1, wherein said exogenous nucleic acid sequence consists of a first and a second exogenous nucleic acid sequence, wherein said first exogenous nucleic acid sequence encodes fatty acid aldehyde acyl-ACP reductase and said second exogenous nucleic acid sequence encodes a fatty acid aldehyde decarbonylase.

5. The composition of claim 1, wherein said microorganism produces and/or secretes at least 60% of one or more lipids or hydrocarbons by weight.

6. The composition of claim 1, wherein said gaseous feedstock comprises syngas.

7. The composition of claim 1, further comprising said one or more lipids or hydrocarbons produced by said microorganism.

8. The composition of claim 1, wherein said microorganism is Cupriavidus necator.

9. The composition of claim 8, wherein said microorganism is Cupriavidus necator DSM 531.

10. The composition of claim 1, wherein said intracellular energy carriers comprise Adenosine-5′-triphosphate (ATP).

11. The composition of claim 1, wherein said inorganic electron donor consists of H2 ,and wherein said gaseous feedstock does not comprise syngas.

12. The composition of claim 1, wherein said exogenous nucleic acid sequence consists of an exogenous nucleic acid sequence that encodes a thioesterase.