WO2011156631A2

WO2011156631A2 - Methods and compositions to extract carbon-based products from host cells

Info

Publication number: WO2011156631A2
Application number: PCT/US2011/039846
Authority: WO
Inventors: Dan Eric Robertson; David Arthur Berry; Christian Perry Ridley
Original assignee: Joule Unlimited Technologies, Inc.
Priority date: 2010-06-09
Filing date: 2011-06-09
Publication date: 2011-12-15
Also published as: WO2011156631A3

Abstract

The invention provides methods for extracting carbon-based molecules of interest produced by host cells cultured in a biphasic system. Methods and apparatus for extracting a carbon-based molecule from a host cell both in the presence of and in the absence of a cyclodextrin in a biphasic system is disclosed. The method provides for continuous production of carbon-based molecules of interest by host cells.

Description

METHODS AND COMPOSITIONS TO EXTRACT CARBON-BASED PRODUCTS

FROM HOST CELLS

FIELD OF THE INVENTION

[0001] The present disclosure relates to compositions and chemical extraction processes for extracting carbon-based molecules such as, e.g., alkanes, alkenes, and fatty acid esters from a production host cell and their transport to a solvent layer in a biphasic system.

BACKGROUND OF THE INVENTION

[0002] The removal of biomolecules produced by a cell culture in a cell culture medium is an important area of research in the field of bio fuel production from host organisms. New processes are needed to effectively extract carbon-based products of interest from a culture of host cells capable of producing carbon-based products of interest.

[0003] Cyclodextrins are cyclic oligosaccharides composed of a-(l,4)-linked glucose units. They typically exist as 6-membered (a-cyclodextrins), 7-membered (β-cyclodextrins), or 8-membered rings (γ-cyclodextrins), however, many alternative forms exist, including well-characterized 32-unit rings, and even at least 150-membered cyclic oligosaccharides are also known. Many cyclodextrin derivatives have been prepared, including methylated forms, which can be produced by reacting parent cyclodextrin with methyl halides. Cyclodextrins have amphiphilic properties in that they have a hydrophilic exterior and a hydrophobic interior, allowing them to solubilize non-polar molecules in aqueous environments. Many uses have been found for them, and they are employed widely in the pharmaceutical, food, and cosmetic industries; see, e.g., Del Valle, E.M.M., Process Biochemistry, 39: 1033-1046 (2004).

[0004] Cyclodextrins and their derivatives have been shown to allow rapid equilibration of cholesterol between vesicles (Leventis, R., et al., BiophysicalJournal, 81 :2257-2267 (2001)), and also can remove cholesterol from macrophage foam cells (Atger, V.M., et al., J. Clin. Invest., 99:773-780 (1997)). Addition of cyclodextrins to hydrocarbon-degrading bacterial cultures increases the rate of alkane catabolism by increasing the solubility of alkanes in aqueous media (Sivaraman, C, et al., World Journal of Microbiology and

Biotechnology, 26:227-232 (2010)). SUMMARY OF THE INVENTION

[0005] The invention relates to methods and compositions used to extract carbon-based molecules from a cell. Various carbon-based molecules can be extracted from a cell and transferred to a solvent layer in a biphasic system. Cyclodextrin can be employed to aid in the transport between the cell and the solvent layer.

[0006] The invention, therefore, provides for a method of extracting a carbon-based molecule from a cell comprising contacting the cell with a solution, wherein the solution comprises at least one cyclodextrin. In one embodiment of the invention, the cell is a prokaryotic cell.

[0007] In one aspect of this invention, the carbon-based molecule is nonpolar. In another aspect of this invention, the carbon-based molecule is a lipophilic fuel molecule. In one embodiment of this invention, the carbon-based molecule is a long-chain hydrocarbon. The long-chain hydrocarbon of this embodiment can be a long-chain alkane, and also can be a long-chain alkene. In an alternative embodiment, the long-chain alkane is a pentadecane, a hexadecane, or a heptadecane.

[0008] In another embodiment of this invention, the carbon-based molecule is an alkane, an alkene, or a fatty acid ester. In an alternative embodiment of the invention, the carbon- based molecule is a 1-nonadecene, a pentadecane, a hexadecane, a heptadecane, or a fatty acid butyl ester. In some aspects, the fatty acid butyl ester is a butyl myristate, a butyl palmitate, a butyl heptadecanoate, a butyl oleate, or a butyl stearate.

[0009] In one aspect, the prokaryotic cell is a photosynthetic organism. In another aspect, the photosynthetic organism is a cyanobacterium. In one embodiment, the prokaryotic cell has at least one genetic modification. In another embodiment, the genetic modification reduces the S-layer of the prokaryotic cell. In an alternative embodiment, the genetic modification reduces the percent composition of glycocalyx of the prokaryotic cell.

[0010] In some aspects, the prokaryotic cell contains one or more heterologously expressed transporter pathways. Also provided are aspects where the prokaryotic cell has at least one protein that is overexpressed. In one embodiment, these overexpressed proteins are part of the heterologously expressed transporter pathways. In another embodiment, the protein component of the heterologously expressed transporter pathway is involved in the transfer of carbon-based molecules to the outer membrane of the prokaryotic cell. In a further aspect, the expression of the gene encoding the protein component is modulated. In another aspect, the expression of the gene encoding the protein component is increased. [0011] In another aspect, the present invention also provides for the transfer of at least one lipophilic molecule from a prokaryotic cell to a solvent layer. In one embodiment, the method employs biphasic cell culture medium. In one aspect, the biphasic cell culture medium has a non-polar phase comprised of dodecane. In another aspect, the biphasic cell culture medium has a non-polar phase comprising a hydrocarbon, wherein the number of carbon atoms in the hydrocarbon ranges from 6 to 30. In another embodiment, the medium comprises at least one antioxidant. In a further embodiment, the antioxidant comprises a butylated hydroxytoluene. In yet another embodiment, the medium comprises at least one internal standard. In another embodiment, the internal standard comprises a heptacosane. In one aspect, the biphasic solution has a pH of less than 8. In another aspect, the biphasic solution has a pH of less than 6. In yet another aspect, the biphasic solution has a pH of less than 4.

[0012] In one embodiment, the cyclodextrin of the present invention can be a a- cyclodextrin, a β-cyclodextrin, or a γ-cyclodextrin. In some embodiments, the cyclodextrin has a modification. In a further embodiment, the cyclodextrin is methylated. In another embodiment, the cyclodextrin is methyl- β-cyclodextrin.

[0013] In one aspect, the presence of the cyclodextrin in the biphasic solution increases the transfer rate of said carbon-based products to solvent phase of said biphasic solution relative to the transfer rate observed using an otherwise identical biphasic solution lacking said cyclodextrin. In a further aspect, the carbon-based product is extracted from the solvent layer.

[0014] In another embodiment, the invention provides for a method wherein the cyclodextrin increases the transfer of a carbon-based product to a nonpolar phase of a biphasic solution such as, e.g., a cell culture medium. In one embodiment, the carbon-based products are removed from a solution. In another embodiment, the invention provides for a method of culturing a prokaryotic cell for the production of a carbon-based molecule with the steps of: culturing the prokaryotic cell in a biphasic medium comprising at least one cyclodextrin, wherein the prokaryotic cell produces the carbon-based molecule; and collecting the carbon-based molecule from the nonpolar phase of the biphasic medium. In another aspect of the invention, the steps of culturing of the prokaryotic cells in a biphasic medium comprising at least one cyclodextrin and collection of the carbon-based molecule from the nonpolar phase are performed repeatedly. This aspect of the invention allows for a continuous culture of the prokaryotic cells and subsequent repeated production and collection of the carbon-based molecule produced by the prokaryotic cells. [0015] The present invention also provides for a composition useful for extracting a carbon-based molecule from a cell, wherein the composition comprises a prokaryotic cell, and a biphasic medium containing at least one cyclodextrin. In another embodiment, the prokaryotic cell of the composition is genetically modified. In another embodiment, the prokaryotic cell produces a carbon-based molecule. In another embodiment, the carbon- based molecule is an alkane, an alkene, or a fatty acid ester.

[0016] In one embodiment a method for extracting a non-polar carbon-based molecule of interest from a photosynthetic microbe is provided, comprising culturing a photosynthetic microbe in a biphasic system, and collecting the non-polar carbon-based molecule of interest from the non-polar phase of the biphasic system.

DRAWINGS

[0017] Figure 1 is a graph showing the accumulation of alkanes in dodecane overlay of JCC1469 cultures. CD = cyclodextrin. Me = methyl.

[0018] Figure 2 is a set of graphs showing JCC1469 cell-associated alkane content over time. (A) a-CD-containing cultures and control culture without CDs. (B) β-CD-containing cultures and control culture without CDs. (C) Me-P-CD-containing cultures and control culture without CDs. CD = cyclodextrin, DD = dodecane, Me = methyl.

[0019] Figure 3 shows JCC1469 culture cell densities after cyclodextrins and/or dodecane were added. (A) a-CD-containing cultures and control culture without CDs. (B) β- CD-containing cultures and control culture without CDs. (C) Me-P-CD-containing cultures and control culture without CDs. CD = cyclodextrin, DD = dodecane, Me = methyl.

[0020] Figure 4 shows 1-nonadecene concentrations of JCC138 cell pellets and medium (as judged by isooctane partitioning of culture supernatant). The error bars are the standard deviation of duplicate samples

DETAILED DESCRIPTION OF THE INVENTION

[0021] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "comprising a cell" includes one or a plurality of such cells, and reference to "comprising the thioesterase" includes reference to one or more thioesterase peptides and equivalents thereof known to those of ordinary skill in the art, and so forth. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0022] Unless otherwise defined herein, scientific and technical terms used in connection with the invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the invention are generally performed according to conventional methods well known in the art and as described in various general and references that are cited and discussed throughout the present specification unless otherwise more specific indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol. I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol. II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0023] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0024] The term "cyclodextrin" as used herein refers to a molecule comprising at least five linked glucopyranose units. Typical cyclodextrins comprise glycopyranose units linked via 1→4 glycosidic linkages. Unmodified cyclodextrins can be cone shaped with an apolar interior and a hydrophilic exterior. Cyclodextrins can be used in an embodiment of the invention to temporarily entrap and transport a carbon-based product of interest.

Cyclodextrins can be chemically modified to provide desired characteristics. These modifications include, for example, methylation, alkylation, hydroxyalkylation,

carboxyalkylation, and addition of an amino, thio, tosyl, glycosyl, or matlosyl group.

Additionally, ethers, esters, anhydro, deoxy, acidic, and basic derivatives can be prepared by chemical or enzymatic reactions. [0025] The term "a-cyclodextrin" or "a-CD" as stated herein refers to a molecule comprising six glucopyranose units. This molecule is also known as Schardinger's a-dextrin, cyclomaltohexaose, cyclohexaglucan, cyclohexaamylose, aCD, ACD, and C6A.

[0026] The term "β-cyclodextrin" or "β-CD" as stated herein refers to a molecule comprising seven glucopyranose units. This molecule is also known as Schardinger's β- dextrin, cyclomaltoheptaose, cycloheptaglucan, cycloheptaamylose, CD, BCD, and C7A.

[0027] The term "γ-cyclodextrin" or "γ-CD" as stated herein refers to a molecule comprising eight glucopyranose units. This molecule is also known as Schardinger's γ- dextrin, cyclomaltooctaose, cyclooctaglucan, cyclooctaamylose, yCD, GCD, and C8A.

[0028] The term "guest molecule" refers to a molecule which is transiently complexed with a host molecule, such as cyclodextrin, in a unique structural relationship by forces other than those of full covalent bonds. In one embodiment of the invention, the guest molecule is the carbon-based product of interest, such as a hydrocarbon, produced by the host cell, and the host molecule is a cyclodextrin, which is capable of transporting the guest molecule in the polar phase to the non-polar phase.

[0029] The term "triphasic culture" as used herein describes a culture system comprising at least a polar and a nonpolar phase, wherein the phases can spontaneously separate from each other or can do so following a centrifugation step. The polar phase can comprise the medium for growth of a host cell culture. The polar phase can comprise an amphiphilic molecule. The nonpolar phase can comprise at least one hydrophobic or amphiphilic molecule useful for increasing production and/or collection of carbon-based products of interest that are released by a host cell. In another embodiment, the nonpolar phase is useful as a solvent for carbon-based products of interest.

[0030] The term "continuous culture" as used herein describes a culture of

microorganisms in a liquid medium which grows steadily for an extended period of time.

[0031] The term "non-polar phase" as used herein refers to a phase comprised of molecules which exhibit a more hydrophobic nature than those in the polar phase of a biphasic solution. Examples of non-polar phases include, but are not limited to nonpolar, polar aprotic, and polar protic solvents. In one embodiment, the non-polar phase comprises hydrocarbons, alcohols, esters, alkanes, alkenes, and/or fatty acids. In another embodiment, the non-polar phase comprises molecules such as, e.g., pentane, cyclopentane, hexane, cyclohexene benzene, cyclohexene, petroleum ether, heptanes, toluene, 1 ,4-dioxane, chloroform, diethyl amine, diethyl ether, triethyl amine, tert-butyl methyl ether,

dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, formic acid, methyl isobutyl ketone, isobutyl alcohol, diethyl ketone, methyl ethyl ketone, n-butanol, isoamyl alchohol, isopropanol, tert-butyl alcohol, n-octanol, p-xylene, m-xylene, n-propanol, ethanol, methanol, acetone, acetic acid, dimethoxy ethane, benzene, butyl acetate, 1-chlorobutane, tetrahydrofuran, ethyl acetate, o-xylene,

hexamethylphosphorus triamide, water, 2-ethoxyethyl ether, N,N-dimethylacetamide, diethylene glycol dimethyl ether, Ν,Ν-dimethylformamide, 2-methoxyethanol, pyridine, propanoic acid, 2-methoxyethyl acetate, benzonitrile, l-methyl-2-pyrrolidinone,

hexamethylphosphoramide, 1 ,4-dioxane, acetic anhydride, dimethyl sulfoxide,

chlorobenzene, deuterium oxide, ethylene glycol, diethylene glycol, propylene carbonate, formic acid, 1 ,2-dichloroethane, glycerin, carbon disulfide, 1 ,2-dichlorobenzene, methylene chloride, nitromethane, 2,2,2-trifluoroethanol, 1 , 1 ,2-trichlorotrifluoroethane, carbon tetrachloride, and/or tetrachloroethylene. In another embodiment, the non-polar phase comprises dodecane. In another alternative embodiment, the non-polar solvent comprises at least one of the group consisting of dodecane, hexadecane, di-2-ethylhexylphthalate-BEHP, and Fluorinert^® FC-70. The non-polar phase can optionally include a surfactant such as, e.g., sodium dodecylsulphate. In some embodiments of the invention, the carbon-based product of interest is soluble in the non-polar phase. In other embodiments of the invention, the non- polar phase comprises the carbon-based product of interest. In another embodiment of the invention, the organic solvents in the non-polar phase comprise hexanes, octanes, decanes and/or dodecanes. In yet another embodiment, the alkanes comprise either or both linear and branching forms. In still another embodiment, the non-polar phase comprises a hydrocarbon of both linear and branching forms. In one aspect of the invention, the hydrocarbon comprises a chain of between 6 and 30 carbon molecules. In one other embodiment of the invention, the non-polar phase is selected based on the temperature of the biphasic culture so as to minimize evaporation of the non-polar phase under the culture conditions. In another embodiment, the non-polar phase is selected for its involvement in the extraction of carbon- based molecules of interest from the host cell.

[0032] The term "polar phase" as used herein refers to a phase comprised of molecules which exhibit a more hydrophilic nature than those in the non-polar phase of a biphasic solution. In one embodiment of the invention, the polar phase comprises the cell culture medium. In another embodiment of the invention, the polar phase further comprises cyclodextrins.

[0033] The term "modified host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term

"modified host cell" as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. The term "host cell" comprises, but is not limited to the intended definition of the term "modified host cell".

[0034] As used herein, the term "molecule" means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., whether naturally occurring or synthetically produced.

[0035] The term "biomass" refers to biological material produced by a biological system including material useful as a renewable energy source.

[0036] The term "hydrocarbon" generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils.

[0037] The term "biofuel" as used herein refers to any fuel that derives from a biological source. Biofuel refers to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.

[0038] The term "fuel component" refers to any compound or a mixture of compounds that are used to formulate a fuel composition. There are "major fuel components" and "minor fuel components." A major fuel component is present in a fuel composition by at least 50% by volume; and a minor fuel component is present in a fuel composition by less than 50%. Fuel additives are minor fuel components.

[0039] The term "fatty ester" includes any ester made from a fatty acid. The carbon chains in fatty acids can contain any combination of the modifications described herein. For example, the carbon chain can contain one or more points of unsaturation, one or more points of branching, including cyclic branching, and can be engineered to be short or long. Any alcohol can be used to form fatty acid esters, for example alcohols derived from the fatty acid biosynthetic pathway, alcohols produced by the production host through non-fatty acid biosynthetic pathways, and alcohols that are supplied in the fermentation broth. [0040] The term "fatty acid" refers to products or derivatives thereof made in part from the fatty acid biosynthetic pathway of the host organism. The fatty acid biosynthetic pathway includes fatty acid synthase enzymes which can be engineered as described herein to produce fatty acid derivatives, and in some examples can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics. Exemplary fatty acid derivatives include for example, short and long chain alcohols, hydrocarbons, and fatty acid esters including waxes.

[0041] The term "carbon-based products of interest" includes alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as

terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly- beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA),

3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate, 1,3 -butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3- hydroxypropionic acid (HP A), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; pharmaceuticals and pharmaceutical intermediates such as 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids, olefins, alkenes and other such suitable products of interest. Such products are useful in the context of fuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, neutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals.

[0042] The term "catabolic" and "catabolism" as used herein refers to the process of molecule breakdown or degradation of large molecules into smaller molecules. Catabolic or catabolism refers to a specific reaction pathway wherein the molecule breakdown occurs through a single catalytic component or a multitude thereof or a general, whole cell process wherein the molecule breakdown occurs using more than one specified reaction pathway and a multitude of catalytic components.

[0043] The term "anabolic" and "anabolism" as used herein refers to the process of chemical construction of small molecules into larger molecules. Anabolic refers to a specific reaction pathway wherein the molecule construction occurs through a single catalytic component or a multitude thereof or a general whole cell process wherein the molecule construction occurs using more than one specified reaction pathway and a multitude of catalytic components.

[0044] The term "endogenous" as used herein with reference to a nucleic acid molecule and a particular cell or microorganism refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell (or its progenitors) using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A gene is still considered endogenous if the control sequences, such as a promoter or enhancer sequences that activate transcription or translation have been altered through recombinant techniques.

[0045] The term "exogenous" as used herein with reference to a nucleic acid molecule and a particular cell or microorganism refers to a nucleic acid sequence or peptide that was not present in the cell when the cell was originally isolated from nature. For example, a nucleic acid that originated in a different microorganism and was engineered into an alternate cell using recombinant DNA techniques or other methods for delivering said nucleic acid is exogenous.

[0046] The term "release" as used herein refers to the movement of a compound from inside a cell (intracellular) to outside a cell (extracellular). The movement can be active or passive. When release is active it can be facilitated by one or more transporter peptides and in some examples it can consume energy. When release is passive, it can be through diffusion through the membrane and can be facilitated by continually collecting the desired compound from the extracellular environment, thus promoting further diffusion. Release of a compound can also be accomplished by lysing a cell. Extracellular compounds in the cell media can also affect release of a compound.

[0047] The terms "immiscible" or "immiscibility" refer to the relative inability of a compound to dissolve in water and are defined by the compounds partition coefficient. The partition coefficient, P, is defined as the equilibrium concentration of compound in an organic phase (in a bi-phasic system the organic phase is usually the phase formed by the fatty acid derivative during the production process, however, in some examples an organic phase can be provided (such as a layer of dodecane to facilitate product separation) divided by the concentration at equilibrium in an aqueous phase (i.e., fermentation broth). When describing a two phase system the P is usually discussed in terms of logP. A compound with a logP of 10 would partition 10: 1 to the organic phase, while a compound of logP of 0.1 would partition 10: 1 to the aqueous phase. [0048] The term "suitable fermentation conditions" generally refers to fermentation media and conditions adjustable with, H, temperature, levels of aeration, etc., preferably optimum conditions that allow microorganisms to produce carbon-based products of interest. To determine if culture conditions permit product production, the microorganism can be cultured for about 24 hours to one week after inoculation and a sample can be obtained and analyzed. The cells in the sample or the medium in which the cells are grown are tested for the presence of the desired product.

[0049] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0050] Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Selected or Engineered Microorganisms for the Production of Carbon-Based Products of Interest

[0051] Microorganism: Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

[0052] A variety of host organisms can be transformed to produce a product of interest. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria.

[0053] The host cell can be a Gram-negative bacterial cell or a Gram-positive bacterial cell. A Gram-negative host cell of the invention can be, e.g., Gluconobacter, Rhizobium, Bradyrhizobium, Alcaligenes, Rhodobacter, Rhodococcus. Azospirillum, Rhodospirillum, Sphingomonas, Burkholderia, Desuifomonas, Geospirillum, Succinomonas, Aeromonas, Shewanella, Halochromatium, Citrobacter, Escherichia, Klebsiella, Zymomonas Zymobacter, or Acetobacter. A Gram-positive host cell of the invention can be, e.g., Fibrobacter,

Acidobacter, Bacteroides, Sphingobacterium, Actinomyces, Corynebacterium, Nocardia, Rhodococcus, Propionibacterium, Bifidobacterium, Bacillus, Geobacillus, Paenibacillus, Sulfobacillus, Clostridium, Anaerobacter, Eubacterium, Streptococcus, Lactobacillus, Leuconostoc, Enterococcus, Lactococcus, Thermobifida, Cellulomonas, or Sarcina.

[0054] Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include

hyperthermophiles, which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococcus lividis; mesophiles, which grow between 15-60°C and psychrophiles, which grow at or below 15°C such as Psychrobacter and some insects. Radiation-tolerant organisms include Deinococcus radiodurans. Pressure- tolerant organisms include piezophiles or barophiles, which tolerate pressure of 130 MPa. Hypergravity- (e.g., >lg) hypogravity- (e.g., <lg) tolerant organisms are also contemplated. Vacuumtolerant organisms include tardigrades, insects, microbes and seeds. Dessicant- tolerant and anhydrobiotic organisms include xerophiles such as Artemia salina; nematodes, microbes, fungi and lichens. Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina. pH-tolerant organisms include alkaliphiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH > 9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH). Anaerobes, which cannot tolerate 0₂ such as Methanococcus jannaschii; microaerophils, which tolerate some 0₂ such as

Clostridium and aerobes, which require 0₂ are also contemplated. Gas-tolerant organisms, which tolerate pure C0₂ include Cyanidium caldarium and metal-tolerant organisms include metalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New York: Plenum (1998) and Seckbach, J. "Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions." In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Origins and the Search for Life in the Universe, p. 511. Milan: Editrice Compositori (1997).

[0055] Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.

[0056] Algae and cyanobacteria include but are not limited to the following genera: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira,

Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritr actus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema,

Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos,

Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella,

Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,

Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,

Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella,

Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,

Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum,

Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron,

Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium,

Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron,

Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,

Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,

Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia,

Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon,

Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia,

Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus,

Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis,

Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,

Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium,

Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate,

Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion,

Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,

Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys,

Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus,

Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria,

Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium,

Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum,

Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis,

Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella,

Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and

Zygonium.

[0057] Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium . [0058] Green sulfur bacteria include but are not limited to the following genera:

Chlorobium, Clathrochloris, and Prosthecochloris .

[0059] Purple sulfur bacteria include but are not limited to the following genera:

AUochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium,

Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis.

[0060] Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila,

Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.

[0061] Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp.,

Thiomicrospira sp., Thiosphaera sp., Thermothrix sp.; obligately chemolithotrophic hydrogen bacteria such as Hydrogenobacter sp., iron and manganese-oxidizing and/or depositing bacteria such as Siderococcus sp., and magnetotactic bacteria such as Aquaspirillum sp.

[0062] Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic sulfur-metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.

[0063] Hyperphotosynthetic conversion requires extensive genetic modification; thus, in some embodiments the parental photoautotrophic organism can be transformed with exogenous DNA.

[0064] Organisms for hyperphotosynthetic conversion include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants), Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp. PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidum, and Chromatium vinosum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus, and

Rhodopseudomonas palusris (purple non-sulfur bacteria). [0065] Yet other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862.

[0066] Still, other suitable organisms include microorganisms that can be engineered to fix carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.

[0067] A common theme in selecting or engineering a suitable organism is autotrophic fixation of C0₂ to products. This covers photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of C0₂ fixation; Calvin cycle, acetyl-CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups ofprokaryotes. The C0₂ fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. Fuchs, G. 1989. Alternative pathways of autotrophic C0₂ fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer- Verlag, Berlin, Germany. The reductive pentose phosphate cycle

(Calvin-Bassham-Benson cycle) represents the C0₂ fixation pathway in many aerobic autotrophic bacteria, for example, cyanobacteria.

Methods for Host Cell Culture

[0068] Suitable fermentation conditions for culturing host cells are provided. In one embodiment of the invention, JB 2.1 medium, as described in PCT application

US2009/006516, herein incorporated by reference in its entirety, is used as a suitable medium for host cell culture. In another embodiment, additional compounds in the JB 2.1 medium can be added, removed, or altered to provide desired effects on the culture. Alternative medium for culturing host cells can be used to provide optimal conditions for the strain and for the desired effect. These alternative mediums are well known in the art. In one embodiment, the medium further comprises a non-polar phase in a biphasic system.

[0069] In one embodiment of the host cell culture, cells are cultured in a biphasic system. In a further embodiment of the biphasic system, the polar phase is comprised of the growth medium for the host cell, and the non- polar phase is comprised of a phase of hydrophobic molecules. The hydrophobic molecules can be hydrocarbons. In one embodiment, the non- polar phase is useful for extracting or collecting or accumulating a carbon-based product of interest. The carbon-based product of interest can be a hydrocarbon. In one embodiment, the non-polar phase is comprised of hydrocarbons. In another embodiment, the the non-polar phase is comprised of a carbon-based product of interest. In yet another embodiment, the non-polar phase comprises a hydrocarbon that is a carbon-based product of interest.

[0070] In another embodiment of the host cell culture, cells are cultured in a biphasic system comprising at least one cyclodextrin. In one embodiment, the cyclodextrin is a α-, β-, or γ-cyclodextrin. In another embodiment, the cyclodextrin is chemically modified. In yet another embodiment, the presence of cyclodextrin increases the probability of transfer of a carbon-based product of interest produced by a host cell to the non-polar phase after release of the carbon-based product of interest by the host cell. In still another embodiment, the presence of cyclodextrin increases the probability of transfer of a hydrocarbon produced by a host cell to the non-polar phase after release of the hydrocarbon by the host cell. In a further embodiment, the cyclodextrin molecule transfers to the non-polar phase a hydrocarbon produced by a cyanobacterium.

[0071] Turbidostats are well known in the art as one form of a continuous culture within which media and nutrients are provided on an uninterrupted basis and allow for non-stop propagation of host cell populations. Turbidostats allow the user to determine information on whole cell propagation and steady-state productivity for a particular biologically -produced end product such as, e.g., host cell doubling time, temporally-delimited biomass production rates for a particular host cell population density, temporally-delimited host cell population density effects on substrate conversion, and net productivity of a host cell substrate conversion of native molecules to carbon based products of interest. Turbidostats can be designed to monitor the partitioning of substrate conversion products to the nonpolar phase or to the liquid or gaseous state. Additionally, quantitative evaluation of net productivity of a carbon-based product of interest can be accurately performed due to the exacting level of control that one skilled in the art has over the operation of the turbidostat. These types of information are useful to assess the parsed and net efficacies of a host cell genetically engineered to produce a specific carbon-based product of interest.

[0072] In one embodiment, identical host cell lines differing only in the nucleic acid and expressed polypeptide sequence of a homologous enzyme are cultured in a uniform- environment turbidostat to determine highest whole cell efficacy for the desired carbon-based product of interest.

[0073] In another embodiment, identical host cell lines differing only in the nucleic acid and expressed polypeptide sequence of a homologous enzyme are cultured in a batch culture or a turbidostat in varying environments (e.g., temperature, pH, salinity, nutrient exposure) to determine highest whole cell efficacy for the desired carbon-based product of interest.

Cyclodextrin

[0074] In one embodiment of the invention, the cyclodextrin molecule consists of six glucopyranose units (a-cyclodextrin), seven glucopyranose units (β-cyclodextrin), or eight glucopyranose units (γ-cyclodextrin). In another embodiment, the cyclodextrin molecule consists of five or more glucopyranose units. In yet another embodiment, the cyclodextrin molecule is in a homogenous mixture of cyclodextrin molecules. In an alternative

embodiment, the cyclodextrin molecule is in a heterologous mixture of cyclodextrin molecules. In a further embodiment, the cyclodextrin molecule consists of five or more a-D- glucopyranoside units linked via a 1→4 glycosidic linkage.

[0075] In the cyclodextrin molecules, each glucopyranose unit has three free hydroxyl groups. One or more of these hydroxyl groups can be modified by substituting the hydrogen atom or the hydroxyl group by a large variety of substituting groups, including, e.g., alkyl, hydroxyalkyl, carboxyalkyl, amino, thio, tosyl, glucosyl, and maltosyl groups. Additionally, ethers, esters, anhydro, deoxy, acidic, basic derivatives can be prepared by chemical or enzymatic reactions. In one embodiment, the cyclodextrin molecule is methylated. In another embodiment, the cyclodextrin molecule is 2-hydroxypropylated.

[0076] Additional modifications can be made to improve solubility of the cyclodextrin derivative. A modification can improve the fitting and/or the association between

cyclodextrin and a desired guest molecule, or possibly stabilize the desired guest molecule, or further can reduce the reactivity and mobility of desired guest molecule. Other modifications to the cyclodextrin can allow for the formation of immobilized cyclodextrin-containing structures or polymers. Also, specific groups, possibly catalytic, can be added to or near the site of target molecule binding.

[0077] The following examples are for illustrative purposes and are not intended to limit the scope of the invention.

EXAMPLE 1

Cyclodextrin-Mediated or Dodecane Phase-Mediated 1-Nonadecene Extraction

[0078] A 5 mL test-tube culture of a spectinomycin-resistant JCC138, a strain derived from wild-type Synechococcus PCC 7002 as described in WO 2009/111513 (herein incorporated by reference in its entirety), in A+/50 mg/L spectinomycin medium was inoculated by scraping an inoculum of cells from a frozen stock into the medium. A+ medium comprises 18.0 g/L sodium chloride, 5.0 g/L magnesium sulfate heptahydrate, 1.0 g/L sodium nitrate, 1.0 g/L Tris, 0.6 g/L potassium chloride, 0.3 g/L calcium chloride (anhydrous), 50 mg/L potassium phosphate monobasic, 34.3 mg/L boric acid, 29.4 mg/L EDTA (disodium salt dihydrate), 3.9 mg/L iron (III) chloride hexahydrate, 4.3 mg/L manganese chloride tetrahydrate, 315.0 μg/L zinc chloride, 30.0 μg/L molybdenum (VI) oxide, 12.2 μg/L cobalt (II) chloride hexahydrate, 10.0 μg/L vitamin Bi₂, and 3.0 μg/L copper (II) sulfate pentahydrate. The culture was incubated in a Multitron II (Infors) shaking incubator (37 °C, 150 rpm, 2 % C0₂/air, continuous light) until it reached an OD₇₃₀ = 7.3. This culture was then added directly to 125 mL flasks containing 30 mL of JB 2.1 medium and 200 mg/L spectinomycin such that all flasks were at an OD₇₃o = 0.1.) Six of these flasks also contained cyclodextrins (Table 1). Five mL of dodecane (Sigma-Aldrich 297879) containing 50 mg/L butylated hydroxytoluene (BHT, Sigma-Aldrich B1378) and 50 mg/L heptacosane (Fluka 51559) was added to each flask. The BHT serves as an antioxidant and the heptacosane serves as an internal standard. These flasks were incubated for seven days in the Infors incubator under the same conditions as for the test tube culture.

[0079] At the end of incubation, water was added to compensate for evaporation loss (based on measured mass loss of flasks from beginning to end of experiment assuming no dodecane evaporated) and OD₇₃₀ for each sample was determined. The solutions in the flasks were poured into 50 mL Falcon tubes and centrifuged using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor at 6,000 rpm for 5 minutes. A volume of 500 μΐ, was removed from the upper dodecane phase and submitted for GC/FID analysis. The remainder of the dodecane and the culture supernatants were discarded and the cell pellets were suspended in 10 mL total volume of water. 100 μΐ, of the cell suspension was removed and pelleted in a microcentrifuge tube. The pellets were suspended in 1 mL of water and pelleted again and the supernatant was discarded as a washing step. An additional centrifugation step was performed and any residual water was removed. The cell pellets were then extracted with approximately 500 μΐ_^ or 1 mL of acetone (Acros Organics 326570010) containing 50 mg/L BHT and heptacosane (tubes were weighed before and after acetone addition allowing the volume of acetone added to each sample to be determined) by vortexing the suspended pellets for 15 s. The tubes were then spun down and the supernatants were submitted for GC/FID analysis.

[0080] The 1-nonadecene concentrations found in the dodecane and cell pellet acetone extracts were quantified using an Agilent 7890A GC/FID equipped with a 7683 series autosampler. One microliter of each sample was injected into the GC inlet under the following conditions: split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min, and temperature: 280° C. The column was an HP-5MS (Agilent, 30 m x 0.25 mm x 0.25μιη), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50 °C, hold one minute; 10°/min increase to 280°C; hold ten minutes. The concentrations of 1-nonadecene found in the extracts were determined based on a calibration curve prepared with authentic 1-nonadecene (Fluka 74230, r.t. 18.8) and normalized to the concentration of the internal standard, heptacosane.

[0081] The % dry cell weight (DCW) of 1-nonadecene found in the dodecane phase and the cell pellets was based on the measurement of OD₇₃o and calculated using the observed average DCW/OD relationship of 0.29 g/L OD (Table 1). Up to 4% of the 1-nonadecene was found in the dodecane in the cyclodextrin-containing cultures where only 0.5% was found in the controls flasks without cyclodextrins added.

Table 1. The cyclodextrin concentration, OD₇₃o, and 1-nonadecene concentration of the JCC138 flask cultures. a-CD = a-cyclodextrin (Sigma C4680), β-CD = β-cyclodextrin (Sigma 4805), Με-β-CD = methyl- β-cyclodextrin (Sigma C4555).

EXAMPLE 2

Cyclodextrin-Mediated or Dodecane Phase-Mediated Alkane Extraction

[0082] A 5 mL test-tube culture of JCC1469 derived from Synechococcus PCC 7002 as described in US. Patent Application No. 12/759,657 (herein incorporated by reference in its entirety) in A+/25 mg/L spectinomycin medium was inoculated by scraping an inoculum of cells from a frozen stock into the medium. The culture was incubated in a Multitron II (Infers) shaking incubator (37 °C, 150 rpm, 2 % C0₂/air, continuous light) until it reached an OD₇₃₀ = 6.7. This culture was then added directly to 125 mL flasks containing 30 mL of JB 2.1 medium and 200 mg/L spectinomycin such that all flasks were at an OD₇₃o = 0.1. Six of these flasks also contained cyclodextrins (Table 2). Approximately 5 mL of dodecane (Sigma-Aldrich 297879) containing 50 mg/L butylated hydroxytoluene (BHT, Sigma-Aldrich B1378) and 50 mg/L heptacosane (Fluka 51559) was added to each flask. Flasks were weighed before and after dodecane addition allowing the volume of dodecane added to each flask to be determined. The BHT serves as an antioxidant and the heptacosane serves as an internal standard. These flasks were incubated for thirteen days in the Infors incubator under the same conditions as for the test tube culture.

[0083] At the end of the experiment, water was added to compensate for evaporation loss (based on measured mass loss of flasks from beginning to end of experiment assuming no dodecane evaporated) and OD₇₃o for each sample was determined. A volume of 500 of the culture was removed from the flasks and pelleted in a microcentrifuge tube. The supernatants were discarded and the cell pellets were suspended in 1 mL of milli-Q water. These cell suspensions were pelleted again, and the supernatant was discarded as a washing step. This water washing step was repeated, following by an additional centrifugation step and removal of any residual water that remained. The cell pellets were then extracted with approximately 500 of acetone (Acros Organics 326570010) containing 50 mg/L BHT and heptacosane (tubes were weighed before and after acetone addition allowing the volume of acetone added to each sample to be determined) by vortexing the suspended pellets for 15 s. The mixtures were then centrifuged in a microcentrifuge at max speed for 2 min to remove particulates and the supernatants were submitted for GC/FID analysis.

[0084] The remaining solutions in the culture flasks were poured into 50 mL Falcon tubes and centrifuged using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (6000 rpm for 5 min). A volume of 500 was removed from the upper dodecane phase and submitted for GC/FID analysis.

[0085] The pentadecane, hexadecane and heptadecane concentrations found in the dodecane and cell pellet acetone extracts were quantified using an Agilent 7890A GC/FID equipped with a 7683 series autosampler. One microliter of each sample was injected into the GC inlet under the following conditions: split 5: 1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m x 0.25 mm x 0.25μιη), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50 °C, hold one minute; 10°/min increase to 280°C; hold ten minutes. The concentrations of the alkanes found in the extracts were determined based on calibration curves prepared with authentic standards (pentadecane, Sigma-Aldrich P3406, retention time (r.t.) 14.6 min; hexadecane, Sigma- Aldrich H6703, r.t. 15.7 min; heptadecane, Sigma-Aldrich, r.t. 16.8 min) and normalized to the concentration of heptacosane (internal standard, r.t. 25.5 min).

[0086] The concentration of the alkanes found in the solvent phase and in the cell pellets is given in Table 2. The concentrations are adjusted to mg/L of the culture so that the concentration of the alkanes can be directly compared in the dodecane phase and in the cells. The concentration of alkanes found in the dodecane phase for those cultures containing cyclodextrins was approximately 2 to 4.5 -fold greater than for those cultures not containing cyclodextrins. The concentration of alkanes found in the cell pellets was also reduced in those cultures which contained cyclodextrins. The total alkanes recovered in both the dodecane phase and in the cell pellet was lower in the cultures which contained

cyclodextrins. This suggests that a substantial amount of the alkanes were extracted from the cells, but not completely transferred to the solvent layer under these culturing and mixing conditions. Out of the total alkanes recovered, as much as 96% were present in the dodecane phase for the cyclodextrin-containing cultures, while less than 10% were present in the dodecane phase for the cultures without cyclodextrin.

Table 2. The cyclodextrin concentration, OD730S, and alkane concentrations of the JCC1469 flask cultures. a-CD = a-cyclodextrin (Sigma C4680), β-CD = β-cyclodextrin (Sigma 4805), Με-β-CD = methyl- β-cyclodextrin (Sigma C4555).

EXAMPLE 3

Cyclodextrin-Mediated or Dodecane Phase-Mediated Fatty Acid Ester and 1-

Nonadecene Extraction

[0087] A 5 mL test-tube culture of JCC1132 in A+ medium containing 100 mg/L spectinomycin was inoculated by scraping an inoculum of cells from a frozen stock into the medium. JCC1132 is a strain of Synechococcus sp. PCC 7002 that has been engineered to produce esters of fatty acids such as found in biodiesel when incubated in the presence of alcohols using standard procedures as described in e.g., WO 2009/111513 (incorporated by reference in its entirety). The culture was incubated in a Multitron II (Infors) shaking incubator (37 °C, 150 rpm, 2 % C0₂/air, continuous light) until it reached an OD₇₃o = 5.4. This culture was then added directly to 125 mL flasks containing 30 mL of JB 2.1 medium, 200 mg/L spectinomycin and 0.05% butanol (vol/vol) such that all flasks were at an OD₇₃o = 0.1. Six of these flasks also contained cyclodextrins (Tables 3 and 4). Five mL of dodecane containing 50 mg/L butylated hydroxytoluene and 50 mg/L ethyl arachidate was added to each flask. The BHT serves as an antioxidant and the ethyl arachidate serves as an internal standard. These flasks were incubated for thirteen days in the Infors incubator under the same conditions as for the test tube culture. An additional 3.8 of butanol was added to each flask at day 2 and day 5.

[0088] At the end of the experiment, water was added to compensate for evaporation loss (based on measured mass loss of flasks from beginning to end of experiment assuming no dodecane evaporated) and OD₇₃₀ for each sample was determined. A volume of 250 of the culture was removed from the flasks and pelleted in a microcentrifuge tube. The supernatants were discarded and the cell pellets were suspended in 500 of milli-Q water. These cell suspensions were pelleted again, and the supernatant was discarded as a washing step. This water washing step was repeated, following by an additional centrifugation step and removal of any residual water that remained. The cell pellets were then extracted with approximately 500 μΐ_^ of acetone containing 50 mg/L BHT and ethyl arachidate (tubes were weighed before and after acetone addition allowing the volume of acetone added to each sample to be determined) by vortexing the suspended pellets for 15 s. The mixtures were then centrifuged in a microcentrifuge at maximum speed for 2 min to remove particulates and the supernatants were submitted for GC/FID analysis.

[0089] The remaining solutions in the culture flasks were poured into 50 mL Falcon tubes and centrifuged using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (6000 rpm for 5 min). A volume of 500 μΐ_^ was removed from the upper dodecane phase and submitted for GC/FID analysis.

[0090] The butyl ester and 1-nonadecene concentrations found in the dodecane and cell pellet acetone extracts were quantified using an Agilent 7890A GC/FID equipped with a 7683 series autosampler. One microliter of each sample was injected into the GC inlet under the following conditions: split 5: 1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min, and a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m x 0.25 mm x 0.25μιη), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50 °C, hold one minute; 10°/min increase to 280°C; hold ten minutes. The concentrations of 1-nonadecene found in the extracts were determined based on a calibration curve prepared with authentic 1-nonadecene (Fluka 74230, r.t. 18.8 min) and normalized to the concentration of ethyl arachidate (internal standard). Butyl myristate (r.t. 19.7 min), butyl palmitate (r.t. 21.5 min), butyl heptadecanoate (r.t. 22.3 min), butyl oleate (r.t. 22.9 min) and butyl stearate (r.t. 23.1 min) were quantified by determining appropriate response factors for the number of carbons present in the butyl esters from commercially- available fatty acid ethyl esters (FAEEs) and fatty acid butyl esters (FABEs). The calibration curves were prepared for ethyl laurate (Sigma 61630), ethyl myristate (Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butyl stearate (Sigma S5001). The concentrations of the butyl esters present in the extracts were determined and normalized to the concentration of ethyl arachidate (internal standard).

[0091] The concentrations of the butyl esters and 1-nonadecene found in the solvent phase and in the cell pellets are given in Table 3 and Table 4. The concentrations are adjusted to mg/L of the culture so that the concentration of the alkanes can be directly compared in the dodecane phase and in the cells. The concentrations of butyl esters found in the dodecane phase for those cultures containing 1 g/L cyclodextrin were approximately 1.5- 12.5 fold greater than for those cultures not containing cyclodextrin. Similarly, the concentrations of 1-nonadecene found in the dodecane phase for those cultures containing cyclodextrins were approximately 2.2-16.2 fold greater than for those cultures not containing cyclodextrins. Out of the total amounts of the respective metabolites that were recovered, up to 49% of the FABE and 23% of 1-nonadecene were found in the dodecane phase from the cyclodextrin-containing cultures, while less than 3% of either metabolite was found as part of the dodecane phase in cultures not containing cyclodextrins.

Cyclodextrin OD₇₃₀ FABEs FABEs Total FABEs % FABEs concentration (mg/L) (mg/L) (mg/L) in

in dodecane in cells dodecane

1 g/L a-CD 6.64 6.58 15.26 21.84 30.1

0.2 g/L a-CD 4.84 4.57 30.18 34.75 13.1

1 g/L β-CD 4.44 9.65 28.98 38.63 25.0

0.2 g/L β-CD 9.36 6.08 55.81 61.90 9.8

1 g/L Me-β- CD 7.80 27.65 28.88 56.53 48.9

0.2 g/L Me-β- CD 7.40 6.07 17.14 23.21 26.2

None 8.08 4.19 229.19 233.38 1.8

None 11.44 2.20 90.84 93.04 2.4

Table 3. The cyclodextrin concentration, OD730S, and butyl esters concentrations of the JCCl 132 flask cultures. FABE = fatty-acid butyl ester, a-CD = a-cyclodextrin, β-CD = β- cyclodextrin, Με-β-CD = methyl^-cyclodextrin.

Table 4. The cyclodextrin and 1-nonadecene concentrations of the JCCl 132 flask cultures. 1- nona = 1-nonadecene, a-CD = a-cyclodextrin, β-CD = β-cyclodextrin, Με-β-CD = methyl- β- cyclodextrin.

EXAMPLE 4

Alkane extraction rates in culture flasks using the cyclodextrin/biphasic system

Strain and Culturing Conditions

[0092] A 5 ml culture JCCl 469 in A+ medium containing 25 mg/L spectinomyin was inoculated from a colony and incubated for 7 days in a Multitron II (Infers) shaking incubator (37 °C, 150 rpm, 2 % C0₂/air, continuous light). This culture was used to inoculate 350 ml of A+ medium containing 200 mg/L spectinomycin such that the flask was at an OD730 = 0.1. This culture was incubated for 18 days. The determined OD730 after sterile milli-Q water back to the flask to compensate for evaporation (based on weight loss of flask over time) was 4.6. 250 μΐ of the culture was removed for acetone cell pellet extraction to determine the intracellular content of alkanes (see Example 2).

[0093] Twenty seven ml of the remaining culture was added to ten 125 ml non-baffled glass flasks, and 3 ml of the lOx concentrated sterile filtered cyclodextrin solutions in A+ medium (or just A+ as a control) was added to yield the JCC1469 cultures shown in Table 5, below:

Table 5

[0094] In addition, five ml of dodecane containing 25 mg/L BHT (antioxidant) and 25 mg/L heptacosane (internal extraction standard) were added to flasks 7-10.

[0095] The flasks were placed incubated in an Infors shaking incubator under the same conditions, and the flasks were sampled at 4, 8, 23, 47, 80, 151 and 198 h. At each timepoint, 500 μΐ of culture was removed from each culture after compensating for water loss due to evaporation. 50 μΐ of the culture was used for OD₇₃o determination, and 450 μΐ was extracted using the acetone method for alkane quantification. 450 μΐ of the dodecane overlay was removed at each timepoint, and replaced with an equal volume so that the total volume of dodecane would not change throughout the experiment.

[0096] GC/FID was used to quantify the pentadecane, hexadecane and heptadecane in the acetone extracts and dodecane samples (see Example 2). The three alkanes concentrations were added to yield the total alkanes value which was converted to mg/L of culture.

Accumulation of alkanes in dodecane over time

[0097] The alkanes accumulated in the dodecane in a linear manner (Figure 1), and the rates of accumulation in the dodecane were 4-1 Ox faster in those flasks which contained 1 g/L of a cylcodextrin (CD) than the control flask which did not contain a CD. The rate of β-CD- mediated accumulation of alkanes increased by a factor of 2x after 80 h whereas the other two extraction rates maintained at the same level throughout the experiment (Table 6).

Table 6

Linear rates of alkane accumulation (mg/L-h) in dodecane phase

in the presence or absence of a cyclodextrin.

methyl, CD = cyclodextrin.

Cell-associated alkane extraction over time

[0098] The cell-associated alkane concentration dropped over time in comparison to the JCC1469 control flask which held relatively constant at around 25 mg/L of culture. β-CD appeared to be a slower extractor of alkanes than the other two tested CD's. The presence of dodecane increased the observed level of extraction for β-CD and a-CD but not Με-β-CD. In the case of Με-β-CD, the presence of dodecane appeared to inhibit the extraction of cells. Therefore, a-CD is superior to the other two cyclodextrins as an extraction catalyst. For all three CDs, the 1 g/L titre of CD extracted more alkanes faster than the 0.5 g/L CD titre (Figure 2). The rates of extraction by the various CDs could not be accurately estimated due to the fact that the cells could have been producing alkanes over the course of the experiment, but the total quantity of alkanes recovered from the CD-containing flasks did not match the control flask (Table 7). This suggests that a significant quantity of alkanes is retained in the CD reservoir or to the glass flask and that the surface area contact of the two phases in this experiment is inadequate to achieve efficient transfer of the alkanes from the aqueous phase to the dodecane phase. Increasing the surface area contact through better mixing of the two phases or employing a different culture apparatus will increase the efficiency of alkane transfer. Table 7

Total alkanes (pentadecane, hexadecane and heptadecane)

in mg/L of culture recovered at 198 h.

CD=cyclodextrin, Me = Methyl, DD = presence of dodecane.

Cell-density and culture health over time

[0099] The optical densities of the cultures after addition of CDs and/or dodecane were relatively consistent through 47 h. By the 80 h timepoint, a larger spread in optical densities in the cultures was apparent. At 151 h, some cylodextrin-containing cultures were at considerably lower ODs than the control culture (Figure 3). This suggests that continuous extraction of cells employing high concentration of CDs may have a negative impact on the cultures after a period of time. All cultures were a healthy-appearing green color from 0 - 80h but had turned brown (presumably chlorotic) by the 151 h timepoint. Using lower concentrations of CDs to extract the alkanes will reduce or eliminate these toxic effects to the cultures.

EXAMPLE 5

pH dependence of cyclodextrin-based extraction method

[0100] Six 3.5 ml replicate JCC138 (i.e., Synechoccocus sp. PCC 7002) cultures at an an OD₇₃₀ = 2.5 were prepared in 15 ml falcon tubes from one culture incubated in A+ medium in a Multitron II (Infers) shaking incubator (37 °C, 150 rpm, 2 % C0₂/air, continuous light). A sample (500 μΐ) of cells was removed from each culture for the acetone cell pellet extraction procedure and quantification of cell-associated 1-nonadecene (see Example 1, above). To duplicate cultures, 30 μΐ of 6N hydrochloric acid (HCl) was added, 30 μΐ of 3M potassium acetate (KOAc) pH 5.5 was added or 30 μΐ of milli-Q water was added. The pH of the cultures was tested by pH paper, and the HCl-treated cultures were found to have a pH = 3 while the KO Ac-treated cultures had a pH = 5.5. The water-treated cultures had a pH = 8 (the pH of A+ medium). 75 mg of Με-β-CD (methyls-CD) was added (25 g/L of CD) to each tube and the cultures were vortexed and inverted until the Me-β CD was in solution. The 3 ml cultures were transferred into culture tubes and incubated for 4 h in an Infors in the conditions described above. Afterwards, one ml of isooctane containing 25 mg/L BHT and 25 mg/L heptacosane was added to each culture and the cultures were vortexed for 20 seconds. After vortexing, 500 μΐ of culture was removed for the acetone cell pellet extraction procedure. The remaining mixture was pelleted and 500 μΐ of the isooctane phase was submitted for GC analysis and quantification of 1-nonadecene.

[0101] After the 4 h extraction, over 90% of the 1-nonadecene had been extracted at pH = 3 and over 75% of the 1-nonadecene was extracted at pH = 5.5. In comparison, only 18% had been extracted at pH = 8 (Table 8). Larger amounts of 1-nonadecene were also recovered from the medium supernatant at lower pH (Figure 4).

Table 8.

The average % of 1-nonadecene remaining in the duplicate cell pellets

after extraction with methyl-P-CD at different pH.

[0102] Cell cultures whose pH has been decreased with HC1, KOAc, or by other means, will be treated to return the pH to normal culture conditions after a specificed amount of time. This process will allow for an increase in the % of 1-nonadecene extracted when compared to cells cultured under constant pH conditions (i.e., where the cell culture pH was not reduced by KOAc, HC1, or by other means). This process will reduce the toxicity of the medium to the cells when compared with keeping the pH at constant levels after a decrease in the pH. This process will be performed a single time, or will be performed multiple times to a culture to enhance cyclodextrin extraction of 1-nonadecene from the JCC138 cells.

[0103] Methods and materials similar or equivalent to those described herein can also be used in the practice of the invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

CLAIMS What is claimed is:

1. A method for extracting a carbon-based molecule from a cell, comprising contacting the cell with a biphasic solution having a solvent layer, wherein said biphasic solution comprises cyclodextrin.

2. The method of claim 1, wherein said carbon-based molecule is non-polar.

3. The method of claim 1, wherein said carbon-based molecule is a lipophilic fuel molecule.

4. The method of claim 1, wherein said carbon-based molecule is a hydrocarbon.

5. The method of claim 4, wherein said hydrocarbon is a long-chain hydrocarbon.

6. The method of claim 5, wherein said long-chain hydrocarbon is a long-chain alkane or an alkene.

7. The method of claim 1, wherein said carbon-based molecule is selected from a group consisting of an alkane, an alkene, and a fatty acid ester.

8. The method of claim 7, wherein said alkane is selected from a group consisting of a pentadecane, a hexadecane, and a heptadecane.

9. The method of claim 1, wherein said carbon-based molecule is selected from the group consisting of a 1-nonadecene, a pentadecane, a hexadecane, a heptadecane, and a fatty acid butyl ester.

10. The method of claim 9, wherein said fatty acid butyl ester is selected from the group consisting of a butyl myristate, a butyl palmitate, a butyl heptadecanoate, a butyl oleate, and a butyl stearate.

11. The method of claim 1 , wherein said carbon-based molecule is a lipophilic fuel molecule.

12. The method of claim 1, wherein said carbon-based molecule is 1-nonadecene.

13. The method of claim 1, wherein said cell is a prokaryotic cell.

14. The method of claim 13, wherein said prokaryotic cell is a photosynthetic cell.

15. The method of claim 14, wherein said photosynthetic cell is a cyanobacterium.

16. The method of claim 1, wherein said cell has at least one genetic modification.

17. The method of claim 15, wherein said cyanobacterium has at least one genetic modification.

18. The method of claim 16, wherein said at least one genetic modification reduces the S- layer of said cyanobacterium.

19. The method of claim 16, wherein said at least one genetic modification reduces the percent composition of glycocalyx of said cyanobacterium.

20. The method of claim 1, wherein said cell contains one or more heterologously expressed transporter pathways.

21. The method of claim 20, wherein said one or more heterologously expressed transporter pathways comprises at least one protein that is over-expressed.

22. The method of claim 20, wherein a protein component of said one or more

heterologously expressed transporter pathways is involved in the transfer of said carbon- based molecules to the outer membrane of said cell, and wherein the expression of the gene encoding said protein is modulated.

23. The method of claim 22, wherein the expression of the gene encoding said protein is increased.

24. The method of claim 1, wherein at least one lipophilic molecule is transferred from said cell to the solvent layer.

25. The method of claim 1, wherein said solvent layer comprises dodecane.

26. The method of claim 1, wherein said solvent layer comprises a hydrocarbon, wherein the number of carbon atoms in said hydrocarbon ranges from 6 to 30.

27. The method of claim 1, wherein said biphasic solution comprises at least one antioxidant.

28. The method of claim 1, wherein said biphasic solution further comprises butylated hydroxytoluene.

29. The method of claim 1, wherein said biphasic solution comprises at least one internal standard.

30. The method of claim 1, wherein said biphasic solution further comprises heptacosane.

31. The method of claim 1 , wherein said cyclodextrin is selected from the group consisting of a-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

32. The method of claim 31 , wherein said at least one cyclodextrin is methylated.

33. The method of claim 32, wherein said at least one cyclodextrin is methyl- β-cyclodextrin.

34. The method of claim 1, wherein the presence of the cyclodextrin in the biphasic solution increases the transfer rate of said carbon-based products to solvent phase of said biphasic solution relative to the transfer rate observed using an otherwise identical biphasic solution lacking said cyclodextrin.

35. The method of claim 1, further comprising extracting the carbon-based product from said solvent layer.

36. The method of claim 1, wherein biphasic solution has a pH of less than 8.

37. The method of claim 1, wherein biphasic solution has a pH of less than 6.

38. The method of claim 1, wherein biphasic solution has a pH of less than 4.

39. A method for culturing a cell for production of a carbon-based molecule comprising the steps of:

a. culturing said prokaryotic cell in a biphasic solution comprising a non-polar phase and a polar phase, and further comprising at least one cyclodextrin, wherein said cell releases said at least one carbon-based molecule, and b. collecting said at least one carbon-based molecule from the non-polar phase of said biphasic solution.

40. The method of claim 39, further comprising at least one repetition of said steps with said cell.

41. The method of claim 39, wherein said method comprises a continuous culture of said cell.

42. A composition for producing carbon-based products, said composition comprising a cell, and a biphasic system containing at least one cyclodextrin.

43. The composition of claim 42, wherein said cell is genetically modified.

44. The composition of claim 42, wherein said cell produces a carbon-based molecule selected from the group consisting of an alkane, an alkene, and a fatty acid ester.

45. A method for extracting a non-polar carbon-based molecule of interest from a

photosynthetic microbe, comprising a) culturing a photosynthetic microbe in a biphasic system, and

b) collecting the non-polar carbon-based molecule of interest from the non-polar phase of the biphasic system.