# Patent

## Title
Production of saturated fatty alcohols from engineered microorganisms

## Abstract

Recombinant bacterial microorganisms are provided which comprise heterologous fatty acyl reductases (“FAR”) polypeptides wherein said microorganisms have been engineered to produce increased amounts of saturated fatty alcohols and methods of making saturated fatty alcohols using the recombinant bacterial microorganisms.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a national stage application filed under 35 USC §371 and claims priority to PCT International Application No. PCT/US2012/069553, filed Dec. 13, 2012, U.S. Provisional Patent Application Ser. No. 61/577,756, filed Dec. 20, 2011, U.S. Provisional Patent Application Ser. No. 61/578,673, filed Dec. 21, 2011, U.S. Provisional Patent Application Ser. No. 61/636,044, filed Apr. 20, 2012, U.S. Provisional Patent Application Ser. No. 61/674,053, filed Jul. 20, 2012, and PCT International Application No. PCT/US2012/069444, filed Dec. 13, 2012. The present application hereby incorporates both of these priority applications by reference, in their entireties and for all purposes.


FIELD OF THE INVENTION
This invention relates to recombinant bacterial microorganisms exhibiting improved properties, especially improved production of saturated fatty alcohols, wherein the fatty alcohol composition comprises fatty alcohols having carbon chain lengths of one or more of C12, C14 and C16.
REFERENCE TO A “SEQUENCE LISTING,” LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file CX5-106WO2_ST25.TXT, created on Dec. 13, 2012, 100,377 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
Crude petroleum has traditionally been used as a primary source for raw materials for producing numerous specialty chemicals. Particular specialty chemicals that can be produced from the petrochemical raw materials include fatty alcohols. Fatty alcohols have many industrial and commercial uses. For example, fatty alcohols act as surfactants which are useful in personal care and household products, such as detergents. Fatty alcohols are also used in waxes, lubricating oils, cosmetics and solvents. The longer chain fatty alcohols may be used for transportation fuels such as diesel and jet fuels. However, obtaining fatty alcohols from crude petroleum requires a significant amount of energy and involves the use of a non-renewable energy source.
Further, even those fatty alcohols that are obtained from renewable sources such as from plant or animal derived fatty acids generally are prepared using a hydrogenation step. Hydrogenation is a costly process step but is utilized to eliminate the double bonds of unsaturated fatty acids. A number of prior art references disclose genetically engineered microorganisms that produce products including fatty acid derivatives such as fatty acid esters and fatty alcohols. For example reference is made to international application publications WO2007/136762; WO2008/119082; WO2010/075483; WO2011/008535 and WO2011/019858; U.S. Pat. No. 6,143,538 and U.S. Pat. No. 8,110,670; and U.S. Patent Pub. Nos. US2012/0115195 and US2012/0142979.
However, a need still exists in the field for improved fatty alcohol production from bioengineered microorganisms that is efficient, cost effective and further tailored for use in particular industrial applications. In addition, for certain industrial applications, it is desirable to have a high degree of saturation because the presence of one or more double bonds in a fatty alcohol may lower the melting point, reduce the shelf-life and/or reduce the heat stability of the compound. Therefore, compositions and methods that provide products having increased saturation levels in fatty alcohols and/or fatty alcohol derivatives are commercially beneficial.
BRIEF SUMMARY OF THE INVENTION
This invention provides improved methods of producing fatty alcohols and fatty alcohol derivative compounds in an engineered host cell. More specifically, the invention provides engineered microorganisms (e.g., bacteria) exhibiting improved properties including the production of fatty alcohols and specifically including the production of saturated fatty alcohols and compositions comprising at least one saturated fatty alcohol (e.g., a composition comprising at least C12, C14, C16 and/or C18 saturated fatty alcohols). In some aspects, the engineered microorganisms include at least one heterologous gene encoding a fatty acyl-ACP reductase enzyme (FAR) and an over-expressed endogenous fatty acid biosynthetic (fab) gene. In some aspects, the engineered microorganisms include at least one heterologous gene encoding a fatty acyl-ACP reductase enzyme (FAR) and an inactivated (e.g. disrupted) gene that confers improved production of saturated fatty alcohols compared to a corresponding microbial organism of the same type in which the gene is not inactivated. In some embodiments, the engineered microorganism is E. coli. 
In one aspect, the invention relates to an engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabZ enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising a plurality of C10 to C18. In some embodiments, the present invention provides compositions comprising a plurality of fatty alcohols selected from C10 to C18 (e.g., C10 to C18; C10 to C16; C12 to C16, or C12 to C14) fatty alcohols. In one embodiment of this aspect, the invention further relates to the engineered bacterial microorganism comprising a second recombinant polynucleotide encoding a FabI enzyme. In another embodiment, the invention further relates to a fatty alcohol composition produced by the engineered microorganisms.
In another aspect the invention relates to an engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabA enzyme wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising at least one C10 to C18 fatty alcohol. In some embodiments, the present invention provides compositions comprising a plurality of fatty alcohols selected from C10 to C18 (e.g., C10 to C18; C10 to C16; C12 to C16, or C12 to C14) fatty alcohols. In some embodiments of this aspect, the invention further relates to the engineered bacterial microorganism comprising a second recombinant polynucleotide encoding a FabI enzyme. In another embodiment of this aspect, the invention further relates to a fatty alcohol composition produced by the engineered microorganisms.
In yet another aspect, the invention relates to an engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and a first recombinant polynucleotide encoding a FabI enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising a plurality of C10 to C18 (e.g., C10 to C18; C10 to C16; C12 to C16, or C12 to C14) fatty alcohols. In some embodiments, the present invention provides compositions comprising a plurality of fatty alcohols selected from C10 to C18 fatty alcohols. In another embodiment, the invention further relates to a fatty alcohol composition produced by the engineered microorganisms.
In additional embodiments, the engineered bacterial microorganisms as described above further comprise a heterologous polynucleotide encoding a thioesterase polypeptide and optionally a polynucleotide encoding a FadD enzyme, wherein the engineered microorganism produces a fatty alcohol composition comprising a plurality of C10 to C18 (e.g., C10 to C18; C10 to C16; C12 to C16, or C12 to C14).
In addition, the invention relates to an engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and an inactivated endogenous fabF gene encoding a FabF enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising at least one C10 to C18 fatty alcohol. In some embodiments, the present invention provides compositions comprising a plurality of fatty alcohols selected from C10 to C18 fatty alcohols. In a one embodiment of this aspect the invention further relates to a fatty alcohol composition produced by the engineered microorganisms.
Furthermore, the invention relates to an engineered bacterial microorganism comprising a polynucleotide encoding a heterologous fatty acyl-ACP reductase enzyme (FAR) and an inactivated endogenous fadR gene encoding a FadR enzyme, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising at least one C10 to C18 fatty alcohol. In some embodiments, the present invention provides compositions comprising a plurality of fatty alcohols selected from C10 to C18 fatty alcohols. In one embodiment of this aspect the invention further relates to a fatty alcohol composition produced by the engineered microorganisms.
In an additional aspect, the invention relates to a method of producing a fatty alcohol composition comprising culturing the engineered microorganisms as described herein under suitable culture conditions in which at least one fatty alcohol is produced. In some embodiments a plurality of fatty alcohols are produced (e.g., C10 to C18; C10 to C16; C12 to C16, or C12 to C14). In some embodiments, the methods further comprise recovering the fatty alcohol(s) from the culture medium and/or cells. In some additional embodiments, the recovered fatty alcohol(s) are provided in a composition. In some embodiments, the composition finds use in any suitable product or method.
In yet an additional aspect, the invention relates to a method for increasing the production of saturated fatty alcohols by engineered microorganisms to provide fatty alcohol compositions comprising the desired levels of saturated fatty alcohols. In some embodiments, the methods involve culturing the engineered microorganisms as described herein under suitable culture conditions to produce a fatty alcohol composition, wherein the fatty alcohol composition comprises at least 60% of saturated C12, C14, C16 and/or C18 fatty alcohols as compared to a fatty alcohol composition comprising C12, C14, C16 and/or C18 fatty alcohols produced by a corresponding bacterial microorganism. In some embodiments, the corresponding bacterial microorganism will be an engineered bacterial microorganism, such as a microorganism that comprises the same heterologous FAR but that does not comprise a heterologous fab gene as encompassed by the invention. In some embodiments, the methods further comprise recovering the fatty alcohol(s) from the culture medium and/or cells. In some additional embodiments, the recovered fatty alcohol(s) are provided in a composition. In some embodiments, the composition finds use in any suitable product or method. In a further aspect, the invention relates to methods of producing fatty alcohol compositions comprising contacting an engineered bacterial microorganism as described herein with at least one fermentable sugar, wherein the fermentable sugar(s) are obtained from or included in a feedstock comprising at least one cellulosic substrate.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the catalytic cycle in E. coli of fatty acid biosynthesis and products derived therefrom. Fatty acid biosynthesis is catalyzed by an enzyme system, which converts malonyl-CoA and acyl-ACP into acyl-ACP species. These enzymes are denoted as FabD; FabH; FabB; FabF; FabG; FabA; FabZ; FabI and ACP and are more fully described below. The enzymes FabB; FabF; FabG; FabA; FabZ; FabI and further ACP are involved in key steps for the elongation of acyl-ACPs in E. coli and for the synthesis of saturated and unsaturated fatty alcohols and fatty acids. Acyl-ACPs can be substrates of thioesterases to produce free fatty acids, which in turn can be converted to acyl-CoA derivatives by acyl-CoA synthetase enzymes (e.g., FadD). The acyl-ACP and acyl-CoA substrates are then enzymatically converted to fatty alcohols by a fatty acyl reductase (FAR) enzyme.
FIG. 2 illustrates key steps for saturated and unsaturated fatty acid biosynthesis in E. coli. Fatty acids are synthesized by the sequential addition of the 2-carbon molecular malonyl-ACP. Fully saturated fatty acids are produced up to a chain length of 8 carbons (C8:0-ACP). In the next elongation step, the isomerase activity of FabA catalyzes the production of cis-Δ3-decenoyl-ACP. This intermediate is extended to C12:1-ACP. The introduced double bond is retained in subsequent elongation steps up to C18:1-ACP. In the parallel saturated pathway trans-Δ3-decenoyl-ACP is reduced by FabI to produce C10:0-acyl-ACP, which can further be elongated to produce predominantly up to C16:0-ACP. The pathways for further elongation steps to, for example, C16:0-acyl-ACP or C18:1-acyl-ACP are not shown.
FIG. 3 describes some polynucleotide and amino acid FAR sequences encompassed by the invention: SEQ ID NO:1 illustrates a codon optimized FAR polynucleotide sequence encoding the wild-type FAR amino acid sequence as set forth in SEQ ID NO:2; SEQ ID NO:3 illustrates a FAR polynucleotide sequence encoding a variant FAR amino acid sequence as set forth in SEQ ID NO:4 (V1); SEQ ID NO:5 illustrates a FAR polynucleotide sequence encoding a variant FAR amino acid sequence as set forth in SEQ ID NO:6 (V2); SEQ ID NO:17 illustrates a FAR polynucleotide sequence encoding the wild-type FAR amino acid sequence as set forth in SEQ ID NO:18; and SEQ ID NO: 27 illustrates a polynucleotide sequence encoding a FAR polynucleotide encoding a variant FAR amino acid sequence as set forth in SEQ ID NO: 28 (V3).



DETAILED DESCRIPTION OF THE INVENTION
This invention provides recombinant bacterial microorganisms, such as E. coli, which have been genetically engineered to express various heterologous fatty acid biosynthetic genes such as fabZ, fabI and/or fabA and preferably express a polynucleotide encoding a heterologous FAR. In some additional embodiments, the present invention provides recombinant bacterial microorganisms that comprise the deletion of one or more endogenous genes to create a desired phenotype. These modified bacterial microorganisms find use in commercial production of fatty alcohols having desired chain length distributions and/or desired saturation levels. Indeed, it is intended that the present invention will find us in the production of fatty alcohols suitable for numerous uses in which certain chain lengths and/or saturation levels are desired.
1. DEFINITIONS
Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in organic chemistry, molecular biology, protein engineering, microbiology, and fermentation science, which are within the skill of the art. Such techniques are well-known and described in numerous texts and reference works well known to those of skill in the art.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Many technical dictionaries are known to those of skill in the art. Although any suitable methods and materials similar or equivalent to those described herein find use in the practice of the present invention, some methods and materials are described herein. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
Also, as used herein, the singular “a”, “an,” and “the” include the plural references, unless the context clearly indicates otherwise. Further, the term “or” is used in the present application to mean the disjunctive “or” and the conjunctive “and”.
Amino acids are designated using the three-letter symbols or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.
Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.
Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.
As used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).
The term “fatty alcohol” as used herein refers to an aliphatic alcohol of the formula R—OH, where the R group is at least 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and more carbons in length. R can be saturated or unsaturated. Further saturated or unsaturated fatty alcohols can be described as “Ca:b-OH”, wherein “a” is an integer that represents the total number of carbon atoms in the fatty alcohol and “b” is an integer that refers to the number of double bonds in the carbon chain. In some embodiments, a fatty alcohol produced according to the methods disclosed herein is a C8-C24 saturated or unsaturated fatty alcohol (i.e., a C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, or C24 fatty alcohol). In some embodiments, multiple fatty alcohols are produced with varying saturation levels. For example, in some embodiments, C10, C12, C14, C16 and/or C18 fatty alcohols are produced. However, it is not intended that the present invention be limited to any particular fatty alcohol nor fatty alcohol saturation level. In some embodiments, one or more of the following fatty alcohols is produced: 1-decanol (C10:0), 1-dodecanol (C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), 1-octadecanol (C18:0).
The abbreviation “ACP” refers to an acyl carrier protein.
The term “fatty acid” as used herein means a compound having the formula RCO2H, wherein R is at least two carbons in length and general R is between 4 and 22 carbons in length. Fatty acids may be saturated or unsaturated and further R can be linear or branched.
The term “fatty acyl-ACP as used herein means a compound of the formula below wherein R is at least three carbons in length and may be a saturated or unsaturated straight or branched chain.




The terms “fatty acyl-CoA reductase”, “fatty acyl reductase”, and “fatty acyl acyl-ACP reductase” (E.C.1.1.1.*) are used interchangeably herein to refer to an enzyme that catalyzes the reduction of a fatty acyl-CoA, a fatty acyl-ACP, or other fatty acyl thioester complex to a fatty alcohol, in a reaction linked to the oxidation of NAD(P)H to NAD(P)+. The abbreviation “FAR” is used herein to refer to these fatty alcohol forming enzymes. In some embodiments, a FAR enzyme includes functional fragments. In some embodiments, the FAR enzyme is a modified or variant FAR, wherein a wild-type FAR has been genetically modified to include at least 1 (at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 or more) amino acid alterations (e.g., substitutions, deletions and/or insertions) as compared to a reference FAR.
The term “acyl-CoA” refers to an acyl thioester formed between the carbonyl carbon of an alkyl chain and the sulfydryl group of the 4′-phosphopantetthionyl moiety of co-enzyme A (CoA) which has the formula R—C(O)—S—CoA, wherein R is an alkyl group having at least 4 carbon atoms and preferably between 10 and 14 carbon atoms. R may be straight or branched and saturated or unsaturated.
“Fatty acid biosynthetic enzymes” comprise a complex of enzymes involved in a number of reactions to produce saturated and unsaturated fatty acids (See, FIGS. 1 and 2). The process is primed by the enzymatic conversion of malonyl-CoA into malonyl-ACP and continues by successive addition of 2 carbons derived from malonyl-ACP residues, providing ACP intermediates (i.e., acyl-ACPs). There are at least 8 enzymes involved fatty acid biosynthesis including FabA, FabB, FabD, FabF, FabG, FabH, FabI, and FabZ, collectively and individually referred to herein as “fatty acid biosynthetic (Fab)” enzymes. Furthermore, the ACP protein plays a key role in fatty acid biosynthesis by anchoring the nascent acyl chain and making the acyl chain accessible to other enzymes.
A “FabZ” enzyme as used herein, refers to a “β-hydroxyacyl-ACP dehydratase” (EC 4.2.1.59 to 4.2.61) that catalyzes the reaction of a (3R)-3-hydroxyacyl-ACP to a transΔ2-enoyl-acylACP+H2O. FabZ is encoded by a fabZ gene.
A “FabA” enzyme as used herein, refers to a “β-hydroxyacyl-ACP dehydratase” (EC 4.2.1.59 to 4.2.61) that catalyzes the reaction of a (3R)-3-hydroxyacyl-ACP to a transΔ2-enoyl-acyl ACP+H2O. In addition, the FabA enzyme as used herein has an isomerase activity which catalyzes the reversible isomerization of a transΔ2-decenoyl-ACP to cisΔ3-decenoyl-ACP. FabA is encoded by a fabA gene.
A “FabI” enzyme as used herein, refers to an enoyl-[ACP] reductase (EC 1.3.1.9 and 1.3.1.10) that catalyzes the reaction of a trans-2,3-dehydroacyl-[ACP]+NAD(P)H+H+ to an acylACP+NAD(P)+. FabI is encoded by a fabI gene.
A “FabF” enzyme as used herein, refers to an 3-oxoacyl-ACP synthase (EC 23.1.170) that catalyzes the reaction of malonly-ACP+a 2,3,4-saturated acyl-ACP to a holo-ACP+a β-ketoacyl-ACP+CO2. A FabF enzyme is encoded by a fabF gene.
A “FadR” protein as used herein, refers to a multifunctional dual regulator that exerts negative control over the fatty acid degradative regulon and activates expression of fabA and fabB. The FadR regulator is encoded by a fadR gene. A “regulon” comprises a set of genes under control of a single regulatory protein. Reference is made to Cronan et al., 1998 Mol. Microbiol. 29:937-943
The term “FadD” enzyme as used herein, refers to an “acyl-CoA synthetase (“ACS”) (EC 6.2.1 (acid-thiol ligases)). In some embodiments, the ACS is classified as EC 6.2.1.3. These ACSs are also known as long chain fatty acid-CoA ligases. An ACS catalyzes the reaction of free fatty acids (both saturated and unsaturated fatty acids) into metabolically active CoA esters (e.g., acyl-CoA) during fatty acid degradation. FadD is encoded by a fadD gene. In some embodiments the FadD may be classified as EC 2.3.1.86 (fatty acyl CoA synthase).
The term “FadK” enzyme as used herein is an acyl-CoA synthetase (ACS) (EC 6.2.1) that catalyzes the reaction of free fatty acids having preferentially C10 or less carbon chain lengths. In some cases the gene (fadK) encoding FadK has also been known as ydiD. Reference is made to Morgan-Kiss R M et al., 2004 J. Biol. Chem., 279:37324-37333.
In some bacterial organisms, (e.g. E. coli) fadD and fadK genes both occur and encode enzymes having ACS activity. In some bacterial organisms there may be more than two genes which encode enzymes having ACS activity.
The term “FadE” enzyme as used herein means an acyl-CoA dehydrogenase enzyme (EC 1.3.99.-). A FadE gene is also known as yafH.
Throughout the specification a reference may be made using an abbreviated gene name or an enzyme name. For example “fadD” refers to a gene encoding an acyl-CoA synthetase enzyme (ACS) or as sometimes referred to herein a FadD enzyme.
The phrase a “fatty alcohol composition” as used herein, means a composition which encompasses at least one fatty alcohol and which is produced from an engineered bacterial microorganisms according to the methods of the invention. The fatty alcohol compositions of the invention may include one or more fatty alcohols. For example a fatty alcohol composition may include only C12 fatty alcohols or a fatty alcohol composition may include for example a combination of C12, C14 and C16 fatty alcohols and these fatty alcohols may be saturated or unsaturated fatty alcohols and they may be linear or branched.
The term “carbon chain length” as used herein means the number of carbon atoms in a carbon chain of a fatty alcohol, fatty alcohol substrate (e.g., fatty acid) or a fatty alcohol derivative. For example the term “C12 fatty alcohol” refers to a fatty alcohol molecule having 12 carbons.
The terms “wild-type” or “native” used in reference to a polypeptide or protein means a polypeptide or protein expressed by a naturally occurring microorganism found in nature. When used in reference to a microorganism, the term means a naturally occurring (not genetically modified or engineered) microorganism.
As used herein, “substrate” refers to a substance or compound that is converted or suitable for conversion into another compound (e.g., a product) by the action of at least one enzyme. The term includes not only a single compound but also combinations comprising more than one compound.
As used herein, “conversion” refers to the enzymatic transformation of a substrate to at least one corresponding product. “Percent conversion” refers to the percent of the substrate that is converted to the product(s) within a specified period of time and under specified conditions.
The term “thioesterase or thioester hydrolase (TE)” enzyme used herein means an enzyme having thioesterase activity. TEs are identified as members of EC 3.1.2.1 to EC 3.1.2.27 and also EC3.1.1.5 and EC 3.1.2.-) and these enzyme which hydrolyze the thioester bond between a carbonyl group and a sulfur atom are classified based on enzyme function and substrate identity. In addition, TEs are classified based on the ThYme database (Thioester-active enzyme). In this classification system, TEs have been classified based on amino acid sequence similarity. Under the ThYme system, TEs are further divided into 24 different families (TE1-TE24). Reference is made to D. C. Cantu et al., (2010) Protein Science, 19:1281-1295 and D. C. Cantu et al., (2011) Nucleic Acid Research 39:doi10:1093/nar/gkq1072. TEs according to the invention will have the ability to catalyze a thioester cleavage reaction hydrolyzing a thioester into an acid and a thiol. TEs useful in the invention may be obtained from a number of sources including plant, bacterial, algal, and fungal sources.
The phrase “preference for cleaving a substrate having a certain carbon chain length” or “predominantly cleaving a substrate having a certain carbon chain length” means that an enzyme cleaves or hydrolyzes mainly substrates having a defined number of carbon atoms. The preference is not necessarily exclusive. For example, an enzyme may have a preference for cleaving substrates with chain lengths of 12 carbons, may still cleave substrates having chain length of 10 or 14 carbon atoms. A more specific non-limiting example includes but is not limited to a TE that predominantly hydrolyzes C12 acyl ACP. The enzyme may still cleave a C10 or C14 ACP substrate.
Nucleic acid sequences may be “introduced” into a cell by protoplast fusion, transfection, transduction, transformation, electroporation or any other suitable method known in the art. A nucleic acid sequence introduced into a eukaryotic or prokaryotic cell may be integrated into a chromosome or may be maintained as an episome.
As used herein, the terms “transformed” and “stably transformed” refer to a cell that has a non-native (i.e., heterologous) polynucleotide sequence integrated into its genome or as an episomal plasmid that is maintained for at least two generations.
As used herein the term “gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
As used herein, an “endogenous” or “homologous” gene refers to a gene that is found in a parental strain of a cell (e.g., a fungal or bacterial cell). As used herein in making comparisons between nucleic acid sequences, “homologous genes” (or “homologue” genes) refers to genes from different, but usually related species, which correspond to each other and are identical or very similar to each other. The term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes).
As used herein, “heterologous” polynucleotides are any polynucleotides that are introduced into a host cell by laboratory techniques, and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.
In some embodiments, when “heterologous” is used with reference to a nucleic acid or polypeptide, the term refers to a sequence that is not normally expressed and secreted by an organism (e.g., a “wild-type” organism). In some embodiments, the term encompasses a sequence that comprises two or more subsequences which are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged in a manner not found in nature (e.g., a nucleic acid open reading frame (ORF) of the invention operatively linked to a promoter sequence inserted into an expression cassette, such as a vector).
As used herein, a “heterologous enzyme” is used in reference to an enzyme that is encoded by a heterologous gene. However, it is also contemplated herein that a heterologous gene can encode an endogenous or homologous enzyme. As used herein, the term “heterologous gene” refers to a gene that occurs in a form not found in a parental strain of the host cell. Thus, in some embodiments, a heterologous gene is a gene that is derived from a species that is different from the species of the host cell expressing the gene. In some embodiments, a heterologous gene is a modified version of a gene that is endogenous to the host cell (e.g., an endogenous gene subjected to manipulation and then introduced or transformed into the host cell). For example, in some embodiments, a heterologous gene has an endogenous coding sequence, but has modifications in the promoter sequence. Similarly, in other embodiments, a heterologous gene encodes the same amino acid sequence as an endogenous gene, but has modifications in codon usage and/or to noncoding regions (e.g., introns), and/or combinations thereof. In some embodiments, the heterologous gene is a gene that has been modified to overexpress a gene product of interest.
As used herein the term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
As used herein, the term “overexpression” refers to any state in which a gene is caused to be expressed at an elevated rate or level as compared to the endogenous expression rate or level for that gene. In some embodiments, “overexpression” includes an elevated translation rate or level of the gene compared to the endogenous translation rate or level for that gene. In some embodiments, overexpression includes an elevated transcription rate or level of the gene compared to the endogenous transcription rate or level for that gene. It is intended that the term encompass overexpression of endogenous, as well as heterologous proteins.
The phrase “altered level of expression” means a polynucleotide or polypeptide in a recombinant microorganism encompassed by the invention is present in an amount or concentration that is different (e.g. greater or less) than the amount or concentration when compared to a corresponding reference microorganism.
As used herein, “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (i.e., non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. “Recombinant,” “engineered,” and “non-naturally occurring,” when used with reference to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (i.e., non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.
As used herein, the term “plasmid” refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some eukaryotes or prokaryotes, or integrates into the host chromosome.
As used herein, “operably linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and/or polypeptide of interest. Thus, a nucleic acid is “operably linked” to another nucleic acid sequence when it is placed into a functional relationship with another nucleic acid sequence.
As used herein, a “control sequence” includes all components, which are necessary and/or advantageous for the expression of a polynucleotide of the present disclosure. Each control sequence may be native or foreign to the polynucleotide of interest. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators.
As used herein, a “modified host cell”, “engineered host cell” or “recombinant host cell” is a cell whose genetic material has been altered using genetic engineering techniques. A genetically modified cell also refers to a derivative of or the progeny of a cell whose genetic material has been altered using genetic engineering techniques. An example of a genetic modification as a result of genetic engineering techniques includes a modification to the genomic DNA. Another example of a genetic modification as a result of genetic engineering techniques includes introduction of a stable heterologous nucleic acid into the cell.
The phrase “a corresponding engineered cell (or microorganism) grown under essentially the same culture conditions” as used herein means a reference host cell (either engineered or native) which is grown under essentially the same culture conditions, including but not limited to pH, temperature, time, and culture media as compared to an engineered cell encompassed by the invention and to which the reference cell is being compared to. In some specific nonlimiting examples the engineered cell encompassed by the invention which comprises heterologous polynucleotides encoding a TE, FAR and FadD will be compared to the “corresponding engineered cell”.
The term “carbon source” as used herein refers to a substrate that is suitable for use as a source of carbon for cell growth.
Nucleic acids “hybridize” when they associate, typically in solution. There are numerous texts and other reference materials that provide details regarding hybridization methods for nucleic acids (See e.g., Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes,” Part 1, Chapter 2, Elsevier, New York, [1993], incorporated herein by reference). For polynucleotides of at least 100 nucleotides in length, low to very high stringency conditions are defined as follows: prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For polynucleotides of at least 200 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at least at 50° C. (“low” stringency), at least at 55° C. (“medium” or “moderate” stringency), at least at 60° C. (“medium-high” stringency), at least at 65° C. (“high” stringency), and at least at 70° C. (“very high” stringency). In some embodiments, the stringency conditions include those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. In other embodiments, the stringency conditions include overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors to accomplish the desired stringency.
As used herein, “naturally-occurring enzyme” refers to an enzyme having the unmodified amino acid sequence identical to that found in nature (i.e., “wild-type”). Naturally occurring enzymes include native enzymes (i.e., those enzymes naturally expressed or found in the particular microorganism).
The term “variant” or “mutant” as used interchangeably herein refer to a polypeptide sequence or polynucleotide sequence encoding a polypeptide, said sequence comprising one or more modifications relative to a corresponding wild-type enzyme (or other specified reference sequence) or the wild-type polynucleotide (or other specified reference sequence) such as substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide. In some embodiments, reference to a variant at an amino acid residue refers to a substitution of the amino acid residue for another amino acid residue. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; U.S. Pat. No. 6,586,182; U.S. Pat. No. 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2):157-78; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423-462; Carter, 1986, “Site-directed mutagenesis,” Biochem. 237:1-7; Minshull, et al., 1999, “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290.
As used herein, the term “isolated” or “recovered” refers to a material that is removed from its original environment (e.g., the natural environment, if it is naturally occurring). For example, the material is said to be “isolated” when it is present in a particular composition in a higher or lower concentration than exists in a naturally-occurring or wild-type organism or in combination with components not normally present upon expression from a naturally-occurring or wild-type organism. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. In some embodiments, such polynucleotides are part of a vector, and/or such polynucleotides or polypeptides are part of a composition, and still considered to be isolated, in that such vector or composition is not part of its natural environment. In some embodiments, the term isolated refers to fatty alcohol compounds of varying chain lengths which are isolated or recovered from an engineered cell according to the invention.
As used herein, the terms “numbered with reference to” or “corresponding to,” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
As used herein, the term “biologically active fragment,” or “functional fragment” refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length FAR of the present invention) and that retains substantially all of the activity of the full-length polypeptide. A biologically active fragment can comprise about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, at about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of a full-length polypeptide to which the functional fragment is being compared (e.g. a functional fragment of a FAR polypeptide may comprise at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98% and even at least 99% of the polypeptide of SEQ ID NOs: 2, 4, 6, or 28).
The term “inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein). Genetic modifications may include but are not limited to complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA or down-regulation of a gene. This can be accomplished, for example, by gene “knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), promoter alteration, or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A deletion may encompass all or part of a gene's coding sequence. The term “knockout” refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene.
As used herein, a “gene deletion” or “deletion mutation” is a mutation in which at least part of a sequence of the DNA making up the gene is missing. Thus, a “deletion” in reference to nucleic acids is a loss or replacement of genetic material resulting in a complete or partial disruption of the sequence of the DNA making up the gene. Any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome. Thus, in some embodiments, the term “deletion” refers to the removal of a gene necessary for encoding a specific protein (e.g., a protease). In this case, the strain having this deletion can be referred to as a “deletion strain.”
With respect to “homologs,” reference to particular gene names is for illustration and not limitation. It is understood that gene names vary from organism to organism and reference to a gene name is not intended to be limiting, but is intended to encompass homologs (or analogous sequences) and polymorphic variants with similar function. In certain embodiments, the analogous or homologous sequence will have at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the named gene or gene product.
“Identity” or “percent identity” in the context of two or more polypeptide sequences refers to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues that are the same. For example, the sequence can have a percent identity of at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% over a specified region to a reference sequence when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms or by manual alignment and visual inspection.
Optimal alignment of sequences for comparison and determination of sequence identity can be determined by a sequence comparison algorithm or by visual inspection (see, generally, Ausubel et al., infra). When optimally aligning sequences and determining sequence identity by visual inspection, percent sequence identity is calculated as the number of residues of the test sequence that are identical to the reference sequence divided by the number of non-gap positions and multiplied by 100. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
An algorithm that may be used to determine whether a polypeptide (such as a FAR or Fab enzyme) has sequence identity to a reference sequence is the BLAST algorithm, which is described in Altschul et al., 1990, J. Mol. Biol. 215:403-410, which is incorporated herein by reference. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915). Other programs that may be used include the Needleman-Wunsch procedure, J. MoI. Biol. 48: 443-453 (1970), using blosum62, a Gap start penalty of 7 and gap extend penalty of 1; and gapped BLAST 2.0 (see Altschul, et al. 1997, Nucleic Acids Res., 25:3389-3402) both available to the public at the National Center for Biotechnology Information Website.
Multiple sequences can be aligned with each other by visual inspection or using a sequence comparison algorithm, such as PSI-BLAST (Altschul, et al., 1997, supra) or “T-Coffee” (Notredame et al., 2000, J. Mol. Bio. 302:205-17). T-Coffee alignments may be carried out using default parameters (T-Coffee Technical Documentation, Version 8.01, July 2009, WorldWideWeb.tcoffee.org), or Protein Align. In Protein Align, alignments are computed by optimizing a function based on residue similarity scores (obtained from applying an amino acid substitution matrix to pairs of aligned residues) and gap penalties. Penalties are imposed for introducing an extending gaps in one sequence with respect to another. The final optimized function value is referred to as the alignment score. When aligning multiple sequences, Protein Align optimizes the “sum of pairs” score, i.e., the sum of all the separate pairwise alignment scores.
As used herein, the term “culturing” refers to growing a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, solid, or semi-solid media).
The term “extracellular environment” means the aqueous solution surrounding a cell membrane, excluding the intracellular space. For example, a secreted enzyme or a compound is found in the extracellular environment. In some embodiments, the extracellular environment comprises the culture medium used to grow the cell.
The term “contacting” refers to combining an enzyme and a substrate under conditions in which the enzyme can act on the substrate. Those skilled in the art will recognize that mixing a solution containing an enzyme with a substrate will effect “contacting.” Similarly, in the context of culturing microorganisms, culturing microorganisms in a media containing a substrate (e.g., a fermentable sugar) will effect “contacting” the microorganism with the substrate.
The term “fermentable sugars” refers to simple sugars (monosaccharides, disaccharides and short oligosaccharides) such as but not limited to glucose, xylose, galactose, arabinose, mannose and sucrose. Fermentable sugar is any sugar that a microorganism can utilize or ferment.
The terms “cleaning compositions” and “cleaning formulations” refer to compositions that find use in the removal of undesired compounds from items to be cleaning, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes, etc.), etc. The terms further refer to any composition that is suited for cleaning, bleaching, disinfecting and/or sterilizing any object and/or surface. It is intended that the terms include, but are not limited to detergent compositions (e.g., laundry and fine fabric detergents), hard surface cleaning formulations (e.g., for glass, wood, ceramics and metal countertops, windows, etc.), oven cleaners, carpet cleaners, fabric fresheners, fabric softeners, hand and machine dish detergents, dish rinse aids, and textile and laundry pre-spotters. In addition, the terms encompass cleaning compositions for use in household and institutional use, including but not limited to liquid cleaning and disinfecting agents, such as anti-bacterial hand soaps and wipes, cleaning bars, mouthwashes, denture cleaners, car shampoos, bathroom cleaners, hair shampoos and conditioners/rinses for humans and other animals, shower gels, foam baths, etc. Indeed, it is not intended that the term be limited to any particular cleaning composition. The terms encompass any materials/compounds selected for the particular type of cleaning compositions desired and the form of the product (e.g., liquid, gel, granule, or spray), as long as the composition is compatible with the fatty alcohol(s) of the present invention. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.
2. FATTY ALCOHOL BIOSYNTHETIC GENES, POLYPEPTIDES AND VARIANTS
FabZ Polypeptides and fabZ Polynucleotides:
In certain embodiments, a polynucleotide sequence encoding a FabZ enzyme has been introduced into an engineered cell (e.g., a bacterial cell). For example, in one embodiment, the polynucleotide sequence encoding a FabZ enzyme is set forth herein as SEQ ID NO:7 and the encoded amino acid sequence is set forth as SEQ ID NO:8. In another embodiment, the polynucleotide sequence encoding a FabZ enzyme is set forth herein as SEQ ID NO:25 and the encoded amino acid sequence is set forth as SEQ ID NO:26. In some embodiments, the FabZ is encoded by a nucleic acid sequence that is at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:7 or SEQ ID NO: 25. In some embodiments, the FabZ is encoded by a nucleic acid sequence that can selectively hybridize to SEQ ID NO:7 or SEQ ID NO: 25 under moderately stringent, stringent or highly stringent conditions, as described hereinabove. FabZ sequences can be identified by any of a variety of methods known in the art. For example, a sequence alignment can be conducted against a database, for example against the NCBI database, and sequences with the lowest HMM E-value can be selected. While both FabA and FabZ enzymes are 3-hydroxyacyl-ACP-dehydrases, the FabZ enzymes lack isomerase activity.
In some embodiments, the FabZ comprises an amino acid sequence that is a least about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to the amino acid sequence set forth in SEQ ID NO:8. In other embodiments, the FabZ comprises an amino acid sequence that is a least about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the FabZ is a functional fragment of a sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% the length of the sequence of SEQ ID NO:8 or SEQ ID NO: 26. In some embodiments, the FabZ is derived from a bacterial species such as E. coli or Bacillus. In other embodiments the FabZ is derived from photosynthetic bacterial such as Rhodobacter (for example, R. capsulatus or R. sphaeroides). In some embodiments, the FabZ is encoded by a Rhodobacter capsulatus fabZ.
Methods to measure the dehydratase activity of FabZ are known to those in the art (See e.g., Heath & Rock, 1996. J. Biol. Chem. 271: 27795-27801).
FabA Polypeptides and fabA Polynucleotides:
In certain embodiments a polynucleotide sequence encoding a FabA enzyme has been introduced into an engineered cell (e.g., a bacterial cell). For example, in one embodiment, the polynucleotide sequence encoding a FabA enzyme is set forth herein as SEQ ID NO:9, and the encoded amino acid sequence is set forth as SEQ ID NO:10. In some embodiments, the FabA is encoded by a nucleic acid sequence that is at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:9. In some embodiments, the FabA is encoded by a nucleic acid sequence that can selectively hybridize to SEQ ID NO:9 under moderately stringent, stringent or highly stringent conditions, as described hereinabove. FabA sequences can be identified by any of a variety of methods known in the art. For example, a sequence alignment can be conducted against a database, for example against the NCBI database, and sequences with the lowest HMM E-value can be selected.
In some embodiments, the FabA comprises an amino acid sequence that is a least about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the FabA is a functional fragment of a sequence having at least about 95% sequence identity to SEQ ID NO:10. In some embodiments, the functional fragment is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% the length of the sequence of SEQ ID NO:10. Methods to measure the dehydratase and isomerase activities of FabA are known (See e.g., Heath & Rock, supra).
FabI Polypeptides and fabI Polynucleotides:
In certain embodiments a polynucleotide sequence encoding a FabI enzyme has been introduced into an engineered cell (e.g., a bacterial cell). For example, in one embodiment, the polynucleotide sequence encoding a FabI enzyme is set forth herein as SEQ ID NO:11, and the encoded amino acid sequence is set forth as SEQ ID NO:12. The FabI (SEQ ID NO:11) and FabI (SEQ ID NO:12) are also referred to herein as “fabI1” and “FabI1.”
In some embodiments, the FabI is encoded by a nucleic acid sequence that is at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:11. In some embodiments, the FabI is encoded by a nucleic acid sequence that can selectively hybridize to SEQ ID NO:11 under moderately stringent, stringent or highly stringent conditions, as described hereinabove. FabI sequences can be identified by any of a variety of methods known in the art. For example, a sequence alignment can be conducted against a database, for example against the NCBI database, and sequences with the lowest HMM.
In some embodiments, the FabI comprises an amino acid sequence that is a least about 80%, about 85%, about 88%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, even about 100% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the FabI is a functional fragment of a sequence having at least about 95% sequence identity to SEQ ID NO:12. In some embodiments, the functional fragment is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, and about 99% the length of the sequence of SEQ ID NO:12. In certain embodiments, the FabI is encoded by a fabI gene found in E. coli, such as GenBank: AAC74370. In some embodiments, the FabI is encoded by a fabI gene found in Rhodobacter capsulatus SB1003 such as GenBank: AD86080 (fabI-1) and in other embodiments such as GenBank: ADE86397 (fabI-2). The percentage of amino acid identity between the E. coli FabI and the R. capsulatus FabI-1 is 49%, and the percent of amino acid identity between E. coli FabI and R. capsulatus FabI-2 is 45.6%. The percent amino acid sequence identity between R. capsulatus FabI-1 and R. capsulatus FabI-2 is 59.2%. Methods for measurement of enoyl-ACP reductase activity are known in the art (See e.g., Heath & Rock, 1995. J. Biol. Chem. 270: 26538-26542).
In certain embodiments, the engineered bacterial cell comprises a) a polynucleotide sequence encoding a FabZ enzyme as described hereinabove, and b) a polynucleotide sequence encoding a FabI enzyme as described herein above. In certain embodiments the FabZ enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:8 and the FabI enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12.
In certain embodiments, the engineered bacterial cell comprises a) a polynucleotide sequence encoding a FabZ enzyme as described hereinabove, and b) a polynucleotide sequence encoding a FabI enzyme as described herein above. In certain embodiments the FabZ enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:26 and the FabI enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12.
In certain embodiments, the engineered bacterial cell comprises a) a polynucleotide sequence encoding a FabA enzyme as described hereinabove and b) a polynucleotide sequence encoding a FabI enzyme as described herein above. In certain embodiments, the FabA enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, and even about 100% sequence identity to SEQ ID NO:10 and the FabI enzyme comprises a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12.
In certain embodiments, the engineered bacterial cell comprises a) a heterologous polynucleotide encoding a FabZ enzyme comprising a polypeptide sequence having at least about 90%, (about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% and even 100%) sequence identity to SEQ ID NO: 8 or SEQ ID NO: 26; b) a heterologous polynucleotide sequence encoding a FabA enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, and even about 100% sequence identity to SEQ ID NO:10 and c) a heterologous polynucleotide encoding a FabI enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12.
3. FATTY ALCOHOL REDUCTASE ENZYMES, POLYNUCLEOTIDES AND VARIANTS
The engineered host cells (e.g., an E. coli strain) encompassed by the invention are modified to express a polynucleotide encoding a heterologous FAR. Polynucleotides encoding FAR enzymes are known in the art (See e.g., WO2011/008535; WO2011/019858; U.S. Ser. No. 13/171,138, US2010/02036; U.S. Pat. No. 7,332,311; U.S. Pat. No. 6,143,538 and Metz et al., 2000, Plant Physiol. 122:635-644).
In some embodiments the acyl-CoA is reduced to a fatty alcohol in a two-step process. An NAD(P)H dependent acyl-CoA reductase converts an acyl-CoA to a fatty aldehyde and then the fatty aldehyde is reduced to a fatty alcohol by a NAD(P)H dependent alcohol dehydrogenase. Enzymes involved in this two-step conversion include the enzymes Acr1 and YqhD. (See, Reiser and Somerville, J. Bacteriol. (1997) 179:2969; Ishige et al., Appl. Environ. Microbiol. (2000) 66:3481; Hofrander et al. (2011) FEBS Letters 585:3538-3543 and Kalscheuer et al., 2006, Appl. Environ. Microbiol. 72:1373).
Preferred fatty alcohol forming acyl-CoA reductases (FARs) useful in the present invention catalyze the direct reduction of acyl-CoA and/or acyl-ACP substrates to fatty alcohols wherein free fatty aldehydes are essentially not released as an intermediate. In effect, these FARs reduce acyl chains to fatty alcohols by one enzymatic step. Depending on the substrate chain length, it is possible to have trace amounts of aldehydes produced and released. In the direct reduction process, FAR converts at least acyl-ACP substrates to a fatty alcohol end-product without the subsequent action of an alcohol dehydrogenase.
While not meant to limit the invention, in some embodiments the FAR is a prokaryotic enzyme. In some embodiments, the FAR is derived from a species of Marinobacter including but not limited to M. algicola, M. alkaliphilus, M. aquaeolei, M. arcticus, M. bryozoorum, M. daepoensis, M. excellens, M. flavimaris, M. guadonensis, M. hydrocarbonoclasticus, M. koreenis, M. lipolyticus, M. litoralis, M. lutaoensis, M. maritimus, M. sediminum, M. squalenivirans, and M. vinifirmus, and equivalent and synonymous species thereof.
In certain embodiments, the FAR is derived from M. algicola strain DG893 and has an amino acid sequence that is at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical and/or at least about 99% identical to SEQ ID NO:2 and/or a functional fragment thereof. In another embodiment, the FAR enzyme has an amino acid sequence that is identical to SEQ ID NO:2. In certain embodiments, the FAR is a variant of the wild-type FAR of SEQ ID NO:2 which has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30 or more amino acid alterations (e.g., substitutions, deletions and/or insertions) relative to SEQ ID NO:2. In certain embodiments, the FAR has an amino acid sequence of at least about 90% (such as at least 93%, at least 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 28. In some embodiments, the variant FAR is FAR-V1 comprising an amino acid sequence of SEQ ID NO: 4 and in other embodiments the variant FAR is FAR-V2 comprising the amino acids sequence of SEQ ID NO: 6 and in further embodiments the variant FAR is FAR-V3 comprising SEQ ID NO: 28. In some embodiments, the variant FAR comprises at least 95% sequence identity to SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 28 and further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more amino acid alterations. Indeed the current invention is not limited to the specifically exemplified FAR variants defined herein above and in the Examples, but include for example the FAR variants described in WO2012/006114, US provisional applications 61/578,673; 61/636,044 and 61/674,053 and PCT International Application No. PCT/US2012/069444. The disclosures of which are hereby specifically incorporated by reference.
In certain embodiments, the FAR is derived from Marinobacter aquaeolei and has an amino acid sequence that is at least about 30% identical, at least about 40%, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical and/or at least about 99% identical to SEQ ED NO:18 and/or a functional fragment thereof. In another specific embodiment, the isolated FAR enzyme has an amino acid sequence that is identical to SEQ ID NO:18. In certain embodiments, the FAR is a variant of the wild-type FAR of SEQ ID NO:18 that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30 or more amino acid alterations (e.g., substitutions, deletions and/or insertions) relative to SEQ ID NO:18.
In other embodiments, the FAR is derived from Marinobacter aquaeolei and has an amino acid sequence that is at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical and/or at least about 99% identical to SEQ ID NO: 5 as disclosed in WO 2012/006114 and/or a functional fragment thereof. In another specific embodiment, the FAR enzyme has an amino acid sequence that is identical to SEQ ID NO: 5. In certain embodiments, the FAR is a variant of the wild-type FAR of SEQ ID NO:5 that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30 or more amino acid alterations (e.g., substitutions, deletions and/or insertions) relative to SEQ ID NO:5. In certain embodiments, the FAR is encoded by a polynucleotide sequence having at least 85% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4 as disclosed in WO 2012/006114.
In other embodiments, the FAR is derived from Marinobacter aquaeolei and has an amino acid sequence that is at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical and/or at least about 99% identical to SEQ ID NO: 1 as disclosed in US 2012/0184006 and/or a functional fragment thereof (such as amino acid residue positions 1 to 364 or amino acid residue positions 365 to 591 of SEQ ID NO: 1). In another specific embodiment, the FAR enzyme has an amino acid sequence that is identical to SEQ ID NO:1 as disclosed in US 2012/0184006. In certain embodiments, the FAR is a variant of the wild-type FAR of SEQ ID NO:1 disclosed in US2012/0184006 that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30 or more amino acid alterations (e.g., substitutions, deletions and/or insertions) relative to SEQ ID NO:1. In certain embodiments, the FAR is encoded by a polynucleotide sequence having at least 85% (at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:2 as disclosed in US 2012/0184006.
In certain embodiments, the FAR is obtained from a marine bacterium selected from the group of Meptuniibacter caesariensis strain MED92, Reinekea sp. strain MED297, Marinomonas sp. strain MED121, unnamed gammaproteobacterium strain HTCC2207, and Marinobacter sp. strain ELB 17, as well as equivalents and synonymous species thereof. In certain embodiments, the FAR is obtained from the genus Oceanobacter. In some embodiments, the FAR is obtained from the Oceanobacter species strain RED65 and has an amino acid sequence that is at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 65%, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, and/or at least about 98% identical to SEQ ID NOs:6 and/or 8 as disclosed in WO 2011/008535. In another specific embodiment, the FAR enzyme for use in the methods disclosed herein comprises or consists of a sequence having about 100% identity to the sequence of SEQ ID NO:6 (“FAR_Ocs”). In other specific embodiments, the isolated FAR enzyme or functional fragment is obtained or derived from Oceanobacter kriegii. In still other specific embodiments, the isolated FAR enzyme or functional fragment is obtained or derived from Oceanobacter strain WH099.
In various embodiments, the FAR is encoded by a polynucleotide selected from the group of FAR Hch (Hahella chejuensis KCTC 2396 GenBank YP_436183.1); FAR_Mac (from marine Actinobacterium strain PHSC20C1); FAR_JVC (JCVI_ORF_1096697648832, GenBank Accession No. EDD40059.1); FAR_Fer (JCVI_SCAF_1101670217388); FAR Key (JCVI_SCAF_1097205236585; FAR_Gal (JCVI_SCAF_1101670289386); Vitis vinifera FAR (GenBank Accession No. CAO22305.1 or CAO67776.1); Desulfatibacillum alkenivorans FAR (GenBank Accession No. NZ_ABII01000018.1); Stigmatella aurantiaca FAR (NZ_AAMD01000005.1); Phytophthora ramorum FAR (GenBank Accession No.: AAQX01001105.1); GenBank Accession no. AAD38039.1; gi|5020215|gb|AAD38039.1|AF149917_1 acyl CoA reductase [Simmondsia chinensis]; GenBank Accession no. BAC79425.1; gi|33146307|dbj|BAC79425.1|fatty-acyl reductase [Bombyx mori]; GenBank Accession no. DQ446732.1 or NM_115529.1; gi|91806527|gb|DQ446732.1|Arabidopsis thaliana clone pENTR221-At3g44560; gi|18410556|ref|NM_115529.1|; and (GenBank Accession no. EU817405.1; gi|210063138|gb|EU817405.1|Ostrinia scapulalis. 
As indicated herein, “heterologous FAR” encompasses wild-type FARs, variants and functional fragments thereof. In various embodiments, a functional fragment of a full-length wild-type FAR or a variant FAR comprises at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the wild-type or reference amino acid sequence. In certain embodiments, a functional fragment comprises about 75%, about 80%, about 85%, at about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the amino acid sequence of a full-length FAR polypeptide (such as bit not limited to FAR-V1, FAR-V2 or FAR-V3).
In some embodiments, the preferred substrates for the heterologous FAR are fatty acyl-ACP substrates comprising carbon chain lengths of C10 to C20. In certain embodiments, the fatty acyl-ACP substrates comprise carbon chain lengths of C12 to C18, and in other embodiments, the fatty acyl-ACP substrates comprise carbon chain lengths of C12 to C16. In certain embodiments, the substrate comprises a majority of saturated hydrocarbons. In certain embodiments, the substrate pool for the heterologous FAR comprises over about 80% (e.g., 85%, 90%, 92%, 94%, 95%, 96%, 97%, and 98%) C12 to C18 fatty acyl-ACP substrates. In other embodiments, the heterologous FAR catalyzes the reduction of fatty acyl CoA substrates to the corresponding fatty alcohol. In certain embodiments, the fatty acyl CoA substrate pool comprises over about 70% (e.g., about 75%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99%) C10 to C18 fatty acyl CoA substrates, over about 70% (e.g., about 75%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99%) C10 to C16 fatty acyl-CoA substrates; and also over about 70% (e.g., about 75%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99%) C12 to C16 fatty acyl-CoA substrates. In certain embodiments, the substrate pool for the heterologous FAR comprises over about 80% (e.g., about 85%, about 90%, or about 95%) C12 to C18 fatty acyl-CoA substrates. In certain embodiments, the C10 to C18 fatty acyl substrates comprises a majority of saturated hydrocarbons.
In some embodiments, the engineered bacterial microorganisms comprise i) a polynucleotide sequence encoding a heterologous FAR, wherein the heterologous FAR has at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to SEQ ID NO: 28; ii) optionally a first polynucleotide sequence encoding a heterologous FabI, wherein the heterologous FabI has at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to SEQ ID NO:12 and iii) optionally a second polynucleotide sequence encoding a heterologous FabZ, wherein the heterologous FabZ has at least (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99%) sequence identity to SEQ ID NO: 8 or SEQ ID NO: 26. In some embodiments, the engineered bacterial microorganism comprises either the first polynucleotide encoding a heterologous FabI or the second polynucleotide encoding a heterologous FabZ. In some embodiments, the engineered bacterial microorganism includes both the first polynucleotide encoding FabI and the second polynucleotide encoding FabZ.
4. EMBODIMENTS INCLUDING THIOESTERASE
Thioesterase:
According to one embodiment of the invention, a microbial host cell is engineered to express a heterologous thioesterase (“TE”). The thioesterase may be one that preferentially uses C12, C14 or C16 ACPs. Depending on the TE used a homogenous population of fatty alcohols may be produced. For example, if the TE is one that predominantly uses C12 ACPs then the fatty alcohol composition produced by a recombinant microbial cell according to the invention will predominantly comprise fatty alcohols having a carbon chain length of C12. In some embodiments, preferred TEs are those that are classified as TE from the Family TE14 in the ThYme database. These sequences may be downloaded from GenBank and UniProt databases (Nucleic Acid Res 201038:D142-D148).
Some nonlimiting examples of TEs that may be used include the “class I” and “class II” acyl-ACP TE fat genes (e.g. fatA or fatB genes and reference is made to A. Jones et al., 1995, Plant Cell 7:359-371). In particular, FatB are preferred TEs (e.g. plant acyl-ACP TEs) useful in the invention. In some embodiments, the TE may be a bacterial acyl-ACP TE. FatB may be obtained for example from Umbellularia california having Accession number Q41635; and AAA34215; Ulmus americana having Accession number AAB71731, Cuphea hookeriana Accession numbers Q39513; AAC49269; AAC49269; and AAC72881; Cinnamonum camphorum having Accession number Q39473; AAC49151; and acyl-ACP thioesterases from Cuphea palustris (AAC49179; and U.S. Pat. No. 5,955,329). Other TEs include without limitation CnFatB (Cocos nucifera, e.g. JF338903; JF338904 and JF338905); ccFAT (Cinnamomum camphora); pdFat (Parabacteroides distasonis, ATCC 8503); gsFat (Geobacillus sp. Y412MC10); pvFAT (Paenibacillus vortex V453); pm FAT (Parabacteroides merdae ATCC 43184); cvFatB (Cuphea viscosissima, JF338906; JF338907; and JF338908); eoFat (Elaeis oleifera) AAD42220 (Elaeis guineensis) and mlFat (Madhuca longofolia var. latifolia).
In some embodiments, homologous or analogous TE genes will be used for the heterologous expression of a TE enzyme.
It is known that different acyl-ACP TE have different degrees of chain length specificity. In some preferred embodiments, the TE useful in the invention is a TE having a preference for cleaving chain lengths of any one of C12, C14 and/or C16 fatty acids from ACP. In some embodiments, having a preference for cleaving chain lengths of any one of C12, C14 and/or C16 fatty acids from ACP means that the thioester hydrolysis will produce fatty acids having at least 85%, (such as at least 88%, 90%, 93%, 95%, 96% or more) of any one of C12, C14 and/or C16 carbon chain lengths.
In one embodiment, the TE is encoded by a gene comprising the polynucleotide sequence having at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 21. In some embodiments, the TE enzyme will comprise at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polypeptide sequence of SEQ ID NO: 22. In some embodiments, the TE gene will comprise at least 85% sequence identity to the polynucleotide sequence of SEQ ID NO: 21. In some embodiments, the TE enzyme will comprise at least 85% sequence identity to the polypeptide sequence of SEQ ID NO: 22. In some embodiments, the TE gene will comprise at least 97% sequence identity to the polynucleotide sequence of SEQ ID NO: 21. In some embodiments, the TE enzyme will comprise at least 97% sequence identity to the polypeptide sequence of SEQ ID NO: 22. In some embodiments, the TE gene will comprise at least 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 21. In some embodiments, the TE enzyme will comprise at least 99% sequence identity to the polypeptide sequence of SEQ ID NO: 22. In some embodiments, the TE gene will comprise the polynucleotide sequence of SEQ ID NO: 21. In some embodiments, the TE enzyme will comprise the polypeptide sequence of SEQ ID NO: 22. In some embodiments the gene encoding the TE enzyme is derived from Umbelluria californica (California Bay) and in other embodiments the gene encoding the TE enzyme is derived from Cinnamomum camphorum. 
In some embodiments, the TE enzyme will be a functional fragment of a reference (e.g. native) TE, such as a TE having deletions at the N-terminal amino acid positions. In certain embodiments, the functional fragment will comprise at least 95% of the reference enzyme. In certain embodiments, the functional fragment will include a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues. In some embodiments, the TE is a variant enzyme having at least 1, at least 5, at least 10, at least 15 or more amino acid modifications, such as substitutions. Non-limiting examples include the TE FatB genes from California Bay, Cinnamomun camphora, or from various Cuphea species such as those disclosed in WO 2011/008565 and reference is made to SEQ ID NOs. 21, 48, 52, 56, 60, 64, 66, 70, 72, 76, 80, 82, 86, 90, 92, 94, 96 and 100 described therein.
Further acyl-ACP TEs that are useful according to the invention are described in the following references: U.S. Pat. No. 5,344,771; U.S. Pat. No. 5,512,482; U.S. Pat. No. 6,150,512; U.S. Pat. No. 5,723,761; U.S. Pat. No. 5,910,631 and WO2010/075483.
Various assays are known which can be used to test for TE activity in a recombinant microorganism transformed with a vector comprising a polynucleotide encoding a TE according to the invention (See, Voelker and Davies, 1994, J. Bacteriol. 76:7320).
Acyl-CoA Synthetase:
As described above, the term “acyl-CoA synthetase” is used synonymously with ACS, acyl-CoA synthetase and FadD. These enzymes mediate the formation of acyl-CoA esters (see FIG. 1). According to an embodiment of the invention, a microbial host cell is engineered to express a recombinant ACS. ACSs that can be expressed to produce acyl-CoA include the E. coli fadD gene comprising the polynucleotide sequence of SEQ ID NO: 19 which encodes the ACS comprising the polypeptide sequence of SEQ ID NO: 6. In some embodiments, the fadD gene will comprise at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, the ACS enzyme will comprise at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polypeptide sequence of SEQ ID NO: 20. In some embodiments, the fadD gene will comprise at least 85% sequence identity to the polynucleotide sequence of SEQ ED NO: 19. In some embodiments, the ACS enzyme will comprise at least 85% sequence identity to the polypeptide sequence of SEQ ID NO: 20. In some embodiments, the fadD gene will comprise at least 97% sequence identity to the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, the ACS enzyme will comprise at least 97% sequence identity to the polypeptide sequence of SEQ ID NO: 20. In some embodiments, the fadD gene will comprise the polynucleotide sequence of SEQ ID NO: 19. In some embodiments, the ACS enzyme will comprise the polypeptide sequence of SEQ ID NO: 20.
In some embodiments, fadD encodes an ACS variant enzyme having at least 1, at least 5, at least 10, at least 15 or more amino acid modifications, such as substitutions. Non-limiting examples include modifications to the gene encoding the ACS of SEQ ID NO: 20.
In some embodiments, homologous fadD genes will be used for the heterologous expression of an ACS enzyme to produce acyl-CoAs. These fadD genes include without limitation, fadD from Acinetobacter sp. NCBI ID YP_045024; fadD from Haemophilus influenza NCBI ID NP_438551; fadD from Pseudomonas aeruginosa NCBI ID_251989 and 251990; BH3101 from Bacillus halodurans NP_243969; yhfL from Bacillus subtilis NP_388908; and fadD from Rhizobium etli CFN NCBI ID— 533919; fadD from Marinobacter algicola ZP_01892995; fadD from Marinobacter aquaeolei YP_958864; fadD from Mycobacterium tuberculosis NP_215722; fadD15 from Mycobacterium tuberculosis NP 216703; fadD19 from Mycobacterium tuberculosis YP_177983; fadD from Rhodopseudomonas palustris YP_00993712; fadD from Pseudomonas fluorscens PfO-1 YP_350081; fadD from Pseudomonas putida ACC77300; fadK from E. coli strain W ZP_07590374; putative fadK from Salmonella typhimurium LT2 NP 460316; and putative fadK from Thermomonospora fusca YP_290214.
In some embodiments, the engineered bacterial cell according to the invention comprises (i) one or more heterologous polynucleotides encoding a polypeptide selected from a) a FabZ enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:8; b) a FabI enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12; c) a FabA enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99%, and even about 100% sequence identity to SEQ ID NO:10, and d) a FabZ enzyme comprising a polypeptide sequence having at least about 95%, about 96%, about 97%, about 98%, about 99% or even 100%) sequence identity to SEQ ID NO: 26; (ii) a heterologous polynucleotide sequence encoding a TE comprising at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polypeptide sequence of SEQ ID NO: 22 and (iii) optionally a heterologous polynucleotide sequence encoding a FadD enzyme comprising at least 70%, (at least 75%, 80%, 85%, 90%, 93%, 95%, 97%, 99%, and even 100%) sequence identity to the polypeptide sequence of SEQ ID NO: 20.
5. DNA CONSTRUCTS, VECTORS AND TRANSFORMATION
In another aspect, the present invention provides polynucleotides encoding the enzymes as described above (e.g. FAR, Fab, Fad and TE enzymes). The polynucleotide can be a DNA or RNA and can be single-stranded or double-stranded. The polynucleotide can be isolated from a naturally occurring microorganism, or prepared wholly or partially via synthetic means. Indeed, it is intended that the polynucleotides of the present invention be produced using any suitable methods and components as known in the art.
In various aspects of the invention, the availability of a polypeptide sequence of a specific enzyme (such as a FAR, fab, fad or TE enzyme) provides a description of all polynucleotides capable of encoding the polypeptide of the known sequence because of the known correspondence of particular codons and the amino acids they encode. In certain embodiments, the degeneracy of the genetic code is used to produce a large number of polynucleotides that encode the polypeptides described herein. In some embodiments, the polynucleotides encoding the desired enzyme are codon-optimized. In some embodiments, the FAR polynucleotides encoding the FAR enzymes described herein are codon-optimized for expression in a recombinant host cells. In particular embodiments, the polynucleotides that encode the FAR enzymes described herein are codon-optimized for expression in a bacterial host cell. In some embodiments, the polynucleotides are codon-optimized for any desired property.
In certain embodiments, the present disclosure provides a vector or DNA construct comprising a polynucleotide encoding a FAR enzyme as described above, wherein the FAR is produced in a host cell. In some embodiments, the polynucleotide is a codon-optimized polynucleotide, such as a polynucleotide having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 17 and/or SEQ ID NO:27 and further hybridizes with SEQ ED NO:1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 17 and/or SEQ ID NO:27 under medium, medium-high, high or very high stringency conditions.
Any suitable DNA construct and vector finds use in the invention (See e.g., WO 2011/008535 which is here in incorporated by reference). In some embodiments, the polynucleotide encodes for a variant FAR enzyme and the polynucleotide sequence will have at least about 90%, at least about 91%, at least about 92%, at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:3, SEQ ID NO: 5 and/or SEQ ID NO:27. In some other embodiments, the variant FAR encoded by the polynucleotide sequence has at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:4, SEQ ID NO: 6 and/or SEQ ID NO:28.
In other embodiments, a FAR enzyme is encoded by a polynucleotide sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 17 and/or SEQ ID NO:27 and further hybridizes with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 17 and/or SEQ ID NO:27 under medium, medium-high, high or very high stringency conditions.
In some embodiments, the polynucleotide encodes for a FabZ enzyme and the polynucleotide sequence will have at least about 90%, at least about 91%, at least about 92%, at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:7 and/or SEQ ID NO:25. In some embodiments, the polynucleotide encodes for a FabA enzyme and the polynucleotide sequence will have at least about 90%, at least about 91%, at least about 92%, at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:9. In some embodiments, the polynucleotide encodes for a FabI enzyme and the polynucleotide sequence will have at least about 90%, at least about 91%, at least about 92%, at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:11. In some embodiments, the polynucleotide encodes for a TE enzyme and the polynucleotide sequence will have at least about 90%, at least about 91%, at least about 92%, at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or even about 100%) sequence identity to SEQ ID NO:21.
In some embodiments, polynucleotides encoding any of the enzymes as described herein (such as but not limited to FabZ, FabI, FabA, FAR and TE) for expression in the recombinant host cells are operably linked to a promoter, and optionally, to other control sequences.
Suitable promoters include, but are not limited to constitutive promoters, regulated promoters, and inducible promoters. Appropriate promoter sequences can be obtained from genes encoding extracellular or intracellular polypeptides which are either endogenous or heterologous to the host cell. Methods for the isolation, identification and manipulation of promoters of varying strengths are available in or readily adapted from the art. See e.g., Nevoigt et al. (2006) Appl. Environ. Microbiol. 72:5266-5273, the disclosure of which is herein incorporated by reference in its entirety.
In certain embodiments, the DNA constructs, vectors and polynucleotides are suitable for expression of a heterologous FabZ, FabI, FabA, FAR or TE enzyme in bacteria. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to the promoters obtained or derived the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, Bacillus megaterium promoters, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731(1978)), as well as the tac promoter (DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25(1993)). Additional promoters include trp promoter, phage lambda PL, T7 promoter, promoters found at PromEC (http://margalit.huji.ac.il/promec/index.html) and the like. Particularly useful promoters include the Trc promoter (Brosius J. et al., (1985) J. Biol. Chem. 260: 3539-3541). Additional promoters suitable for use in the present disclosure are described in Terpe H., 2006, Appl. Microbiol. Biotechnol. 72:211-222 and in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York. In some instances it may be advantageous to express the heterologous enzymes, such as the FAR and/or FabZ, FabI or FabA at certain stages during the engineered microorganisms growth cycle. In these cases the heterologous gene may be operably linked to an inducible promoter. Inducible promoters are known in the art. The lac, tac and trc promoters mentioned above are inducible promoters which are induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG).
In various embodiments, an expression vector optionally contains a ribosome binding site (RBS) for translation initiation, and a transcription terminator, such as the transcriptional terminators T1 and T2 derived from the rrnB operon from E. coli (See e.g., Orosz et al., (1991) Eur. J. Biochem. 201: 653-659). The vector also optionally includes appropriate sequences for amplifying expression, e.g., translational enhancers.
In various embodiments, the polynucleotides useful for expressing the heterologous enzymes in recombinant host cells are operably linked to other control sequences, including but not limited to, a transcription terminator sequence, a signal sequence that when translated directs the expressed polypeptide into the secretory pathway of the recombinant host cell, and/or a polyadenylation sequence (eukaryotes). The choice of appropriate control sequences for use in the polynucleotide constructs of the present disclosure is within the skill in the art and in various embodiments is dependent on the recombinant host cell used and the desired method of recovering the fatty alcohol compositions produced. Indeed, it is not intended that the present invention be limited to any particular control sequence(s).
A recombinant expression vector according to the invention can be any suitable vector, e.g., a plasmid or a virus, which can be manipulated by recombinant DNA techniques to facilitate expression of at least one heterologous enzyme in the recombinant host cell. In certain embodiments, the expression vector is integrated into the chromosome of the recombinant host cell and comprises one or more heterologous genes operably linked to one or more control sequences useful for production of at least one heterologous enzyme. In other embodiments, the expression vector is an extra chromosomal replicative DNA molecule, e.g., a linear or closed circular plasmid, that is found either in low copy number (e.g., from about 1 to about 10 copies per genome equivalent) or in high copy number (e.g., more than about 10 copies per genome equivalent). In various embodiments, the expression vector includes a selectable marker, such as a gene that confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance) to the recombinant host organism that comprises the vector.
Expression vectors which, in certain embodiments, are useful for expressing enzymes as disclosed herein (for example FabZ, FabI, FAR and TE) are commercially available, e.g., from Sigma-Aldrich Chemicals, St. Louis Mo. and Stratagene, LaJolla Calif. In some embodiments, examples of suitable expression vectors are plasmids which are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201). In some embodiments, the expression vector comprising a polynucleotide sequence encoding the FAR enzyme and the expression vector comprising a polynucleotide sequence encoding one or more other enzymes, such as but not limited to, a Fab enzyme (e.g., FabZ or FabI) are on separate vectors. In other embodiments, the polynucleotide sequence encoding a heterologous FAR enzyme and the polynucleotide sequence encoding one or more other enzymes such as but not limited to a Fab enzyme are encoded on the same expression vector, and expression of each enzyme is may be independently regulated by a different promoter. In some further embodiments, the polynucleotide sequence encoding the heterologous FAR enzyme and the heterologous Fab enzyme are found on the same expression vector, and expression of each enzyme is regulated by the same promoter.
In certain embodiments, the present disclosure provides a plasmid for expression of heterologous genes in E. coli. Expression vector pCK110900, which comprises a P15A origin of replication “ori” (P15A ori), lac a CAP binding site, a lac promoter, a T7 ribosomal binding site (T7g10 RBS) and a chloramphenicol resistance gene (camR) is an exemplary vector that finds use in the present invention. This expression vector is depicted in FIG. 3 of U.S. Patent Publication No. 2006/0195947, which is incorporated herein by reference in its entirety. Other suitable plasmid vectors include, but are not limited to derivatives of pCL1920 and pCL1921 (Lerner and Inouye, 1990; NAR 18:4631). These vectors contain the pSC101 ori and confer resistance to spectinomycin (GenBank:AB236930). In some embodiments, the vector is an expression vector derived from pCL1920 including the Trc promoter and the lacIq gene from E. coli. 
Methods, reagents and tools for transforming host cells described herein, such as bacteria, yeast (including oleaginous yeast) and filamentous fungi are known in the art. General methods, reagents and tools for transforming, e.g., bacteria can be found, for example, in Sambrook et al (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, New York. Methods, reagents and tools for transforming yeast are described in “Guide to Yeast Genetics and Molecular Biology,” C. Guthrie and G. Fink, Eds., Methods in Enzymology 350 (Academic Press, San Diego, 2002). Methods, reagents and tools for transforming Y. lipolytica are found in “Yarrowia lipolytica,” C. Madzak, J. M. Nicaud and C. Gaillardin in “Production of Recombinant Proteins Novel Microbial and Eucaryotic Expression Systems,” G. Gellissen, Ed. 2005. In some embodiments, introduction of the DNA construct or vector of the present invention into a host cell is accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, or other common techniques (See Davis et al., 1986, Basic Methods in Molecular Biology, which is incorporated herein by reference). In one embodiment, a preferred method used to transform E. coli strains is electroporation and reference is made to Dower et al., 1988) NAR 16: 6127-6145. Indeed, any suitable method for transforming host cells finds use in the present invention. It is not intended that the present invention be limited to any particular method for introducing nucleic acids such as constructs into host cells.
The present invention also provides a method for producing a recombinant host cell, comprising: (a) providing at least one nucleic acid construct comprising at least one polynucleotide sequence encoding a) a FabZ, a FabA and/or FabI enzyme operably linked to a promoter; and (b) transforming a host cell with the nucleic acid construct(s) to produce a recombinant cell. In some embodiments, the host cell is a bacterial cell and in other embodiments, the host cell is E. coli. In some embodiments, the host cell already comprises a polynucleotide sequence encoding a heterologous FAR enzyme as described herein (i.e., the host cell comprises the FAR sequence prior to transformation). In some other embodiments, the FAR polynucleotide and the fab genes (e.g. fabZ, fabZ-fabI, fabA or fabA-fabI) will be on the same DNA construct or vector.
6. METHODS FOR GENE INACTIVATION AND EMBODIMENTS INCLUDING INACTIVATED FAB AND FAD GENES
In some embodiments, endogenous genes of the engineered microorganism of the present invention have been inactivated. For example in some embodiments, engineered microorganisms have been genetically modified to at least partially delete a gene encoding an endogenous enzyme (e.g., FabF, FadE or FadR). Typically, these modifications of the gene reduce or eliminate the total amount of endogenous enzyme produced by the host cell. In some embodiments, complete or near-complete deletion of the gene sequence is contemplated. However, a deletion mutation need not completely remove the entire gene sequence encoding the enzyme, in order to reduce the amount of endogenous enzyme produced by the engineered cell. For example, in some embodiments, there is a partial deletion that removes one or more nucleotides encoding a portion of an enzyme that plays a role in endogenous enzyme activity by the host cell (See, U.S. Pat. No. 8,110,670).
A deletion in a gene encoding an enzyme (e.g., FabF, FadE or FadR) in accordance with the embodiments provided herein includes a deletion of one or more nucleotides in the gene encoding the target enzyme. In some embodiments, there is a deletion of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, of the gene (e.g. a gene encoding for example FabF, FadE or FadR), wherein the amount of enzyme produced by the cell is reduced.
Thus, in some embodiments, the deletion results in at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about a 99% reduction in the enzyme activity produced by the cell, relative to the enzyme activity of a corresponding enzyme produced by an unmodified organism grown or cultured under essentially the same culture conditions and including the gene coding for the corresponding enzyme which had not be inactivated or deleted. In some embodiments, deletion is of a fabB gene or fadR gene.
Inactivation, such as a deletion, of a gene of interest (e.g., a fadE, a fabF, a fabB gene or a fadR gene) can be detected and confirmed by any of a variety of methods known in the art, including the methods provided herein. For example, gene deletion can be confirmed using PCR amplification of the modified genomic region. It will be appreciated that additional suitable techniques for confirming deletion can be used and are well known, including but not limited to Southern blot techniques, DNA sequencing of the modified genomic region, and screening for positive or negative markers incorporated during recombination events.
Some additional methods for complete and/or partial deletion of a gene are well-known. The genetically modified cells described herein can be generated using any of a variety of deletion methods known in the art that result in the complete inactivation or at least a reduction in the amount of at least one endogenous gene expressed by the cells. There are numerous approaches to create genetic modifications in bacteria (See e.g., Court et al., (2002) Annual Rev. Genet 36:361-388; and Datsenko and Wanner (2000) PNAS 97:6640-6645).
In certain embodiments the inactivation is of a fabF polynucleotide sequence encoding a FabF enzyme, For example, in one embodiment, the polynucleotide sequence encoding a FabF enzyme is set forth herein as SEQ ID NO: 13 and the encoded amino acid sequence is set forth as SEQ ID NO: 14. In some embodiments, the FabF is encoded by a nucleic acid sequence that is at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:13. In some embodiments, the FabF is encoded by a nucleic acid sequence that can selectively hybridize to SEQ ID NO:13 under moderately stringent or highly stringent conditions, as described hereinabove. In some embodiments, the FabF enzyme has an amino acid sequence that is at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:14. FabF sequences can be identified by any of a variety of methods known in the art. For example, a sequence alignment can be conducted against a database, for example against the NCBI database, and sequences with the lowest HMM E-value can be selected.
In certain embodiments the inactivation is of a fadR polynucleotide sequence encoding a FadR enzyme. For example, in one embodiment, the polynucleotide sequence encoding a FadR enzyme is set forth herein as SEQ ID NO:15, and the encoded amino acid sequence is set forth as SEQ ID NO:16. In some embodiments, the FadR is encoded by a nucleic acid sequence that is at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:15. In some embodiments, the FadR is encoded by a nucleic acid sequence that can selectively hybridize to SEQ ID NO:15 under moderately stringent or highly stringent conditions, as described hereinabove. In some embodiments, the FadR has an amino acid sequence that is at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:16. FadR sequences can be identified by any of a variety of methods known in the art. For example, a sequence alignment can be conducted against a database, for example against the NCBI database, and sequences with the lowest HMM E-value can be selected.
In certain embodiments, an engineered cell of the invention (e.g., E. coli) comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 30% identical, at least about 40%, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NO:2 and/or a functional fragment thereof and an inactivated gene encoding a FabF enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14. In another embodiment, the engineered cell of the invention comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 95% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NOS:4 or 6 or a functional fragment thereof, and an inactivated gene encoding a FabF enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14.
In certain embodiments, an engineered cell of the invention (e.g., E. coli) comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical, at least about 95% identical, at least about 97% identical, at least about 98% identical and/or at least about 99% identical to SEQ ID NO:2, and/or a functional fragment thereof and an inactivated gene encoding a FadR enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:16. In another embodiment, the engineered cell of the invention comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 95% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NOS:4 and/or 6, and/or a functional fragment thereof and an inactivated gene encoding a FadR enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to SEQ ID NO:16.
In certain embodiments, an engineered cell of the invention (e.g., E. coli) comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 30% identical, at least about 40%, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75%, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 93% identical at least about 95% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NO:2 and/or a functional fragment thereof; an inactivated gene encoding a FadR enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:16; and in activated gene encoding a FabF enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14. In another embodiment, the engineered cell of the invention comprises a polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 95% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NOS:4 and/or 6, and/or a functional fragment thereof; an inactivated gene encoding a FadR enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:16; and an inactivated FabF enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14.
In some embodiments, an engineered bacteria microorganism according to the invention encompasses a polynucleotide sequence encoding a heterologous FAR; one or more heterologous polynucleotide sequences encoding i) a FabZ enzyme, ii) a FabI enzyme, and/or iii) a FabA enzyme; and one or more inactivated genes such as a fabF or fadR. gene. For example, in certain embodiments, an engineered bacterial microorganism according to the invention comprises (a) an heterologous FabZ comprising an amino acid sequence that is a least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% identical to the amino acid sequence set forth in SEQ ID NO:8 and/or a functional fragment thereof; and/or (b) an heterologous FabI comprising an amino acid sequence that is a least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% identical to the amino acid sequence set forth in SEQ ID NO:12 and/or a functional fragment thereof; and/or (c) an heterologous FabA comprising an amino acid sequence that is a least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% identical with the amino acid sequence set forth in SEQ ID NO:10 and/or a functional fragment thereof; and (d) an inactivated gene selected from a gene encoding (i) a FadR enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:16; and/or (ii) a FabF enzyme having at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to SEQ ID NO:14; and (e) a heterologous polynucleotide sequence encoding a FAR enzyme comprising an amino acid sequence that is at least about 30% identical, at least about 40%, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least 91% identical, at least 92% identical, at least about 93% identical, at least about 95% identical, at least 96% identical, at least about 97% identical, at least about 98% identical, and/or at least about 99% identical to SEQ ID NO:2.
7. ENGINEERED HOST CELLS
In some embodiments, the engineered host cell is a prokaryotic cell. Suitable prokaryotic cells include Gram-positive, Gram negative and Gram-variable bacterial cells. In certain embodiments, host cells include, but are not limited to, species of a genus selected from the group of Acetobacter, Acinetobacter, Agrobacterium, Arthrobacter, Bacillus, Clostridium, Corynebacterium, Desulfovibrio, Escherichia, Erwinia, Geobacillus, Klebsiella, Lactobacillus, Mycobacterium, Pantoea, Rhodococcus, Rhotobacter, Streptomyces, Vibrio, and Zymomonas. In certain embodiments, the recombinant host cell is an industrial bacterial strain.
Numerous bacterial industrial strains are known and suitable for use in the methods disclosed herein. In some embodiments, the bacterial host cell is a species of the genus Bacillus, e.g., B. thuringiensis, B. anthracis, B. megaterium, B. subtilis, B. lentos, B. circulans, B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, B. subtilis, B. pumilus, and B. amyloliquefaciens. In some embodiments, the bacterial host cell is a species of the genus Erwinia, e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E. punctata and E. terreus. In other embodiments the bacterial host cell is a species of the genus Pantoea, e.g., P. citrea or P. agglomerans. In still other embodiments, the bacterial host cell is a species of the genus Streptomyces, e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus or S. lividans. In further embodiments, the bacterial host cell is a species of the genus Zymomonas, e.g., Z. mobilis or Z. lipolytica. In further embodiments, the bacterial host cell is a species of the genus Rhodococcus, e.g. R. opacus. 
In some embodiments, the bacterial host cell is a species of the genus Escherichia, e.g., E. coli. In various embodiments, the engineered E. coli bacterial strains useful in the processes described herein are derived from strain W3110, strain MG1655, strain B766 (E. coli W) and strain BW25113. In some further embodiments, the W3110 strain finds use in the present invention; the genome of this strain has been fully sequenced and annotated (See e.g., Hayashi et al., (2005) Mol. Syst. Biol. 2:2006.0007). For industrial applications, phage-resistant strains are particularly useful. In this sense, deletion of the fhuA gene (also known as tonA) confers resistance to phages T1, T5 and phi80 (Link et al., 1997, J. Bact. 179: 6228-8237). Another useful strain is E. coli W (Archer et al., 2011, BMC Genomics, 12:9.doi:10.1186/1471-2164-12-9). Also reference is made to Elben et al. (2005) J. of Food Protection 68(2):282-291.
Other examples of useful E. coli strains include, but are not limited to, E. coli strains found in the E. coli Stock Center from Yale University (http://cgsc.biology.yale.edu/index.php); the Keio Collection, available from the National BioResource Project at NBRP E. coli, Microbial Genetics Laboratory, National Institute of Genetics 1111 Yata, Mishima, Shizuoka, 411-8540 Japan (www at shigen.nig.ac.jp/ecoli/strain/top/top.jsp); or strains deposited at the American Type Culture Collection (ATCC).
The disclosed invention further contemplates the use of other organism such as photosynthetic organisms including but not limited to algae (e.g., cyanobacteria (blue green algae) and photosynthetic bacteria. Non-limiting examples include strains of Synechococcus, Synechocystis, Rhodobacter, Rhodococcus, Chlamydomonas, Chlorella, Prototheca, and Cyanobacterium. Additional heterotrophic species which may be used as host strains that are transformed with genes encoding enzymes involved in the synthesis of fatty alcohols include yeast cells such as but not limited to Saccharomyces (e.g., S. cerevisiae), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. troplicalis) and Schizosaccharomyces (e.g., S. pombe).
8. FERMENTATION/CULTURING
Any suitable means for culturing the recombinant host cells finds use in the present invention. Indeed, any suitable fermentation protocol finds use in the production of the fatty alcohols provided herein. In some embodiments, fermentation of the recombinant host cells as described hereinabove comprises fermenting host cells comprising: 1) a heterologous polynucleotide encoding a FabZ and a heterologous polynucleotide encoding a FAR enzyme; 2) a heterologous polynucleotide encoding a FabI and a heterologous polynucleotide encoding a FAR enzyme; 3) a heterologous polynucleotide encoding a FabZ; a heterologous polynucleotide encoding a FabI; and a heterologous polynucleotide encoding a FAR enzyme; 4) a heterologous polynucleotide encoding a FabA and a heterologous polynucleotide encoding a FAR enzyme; 5) a heterologous polynucleotide encoding a FabA; a heterologous polynucleotide encoding a FabI; and a heterologous polynucleotide encoding a FAR enzyme; 6) a heterologous polynucleotide encoding a FAR enzyme and an inactivated fabF encoding a FabF polypeptide; or 7) a heterologous polynucleotide encoding a FAR enzyme and an inactivated fadR encoding a FadR polypeptide; under suitable conditions and for a time sufficient for production of fatty alcohols, as desired. Conditions for the culture and production of cells, including bacterial and yeast cells, are readily available and well-known in the art. Cell culture media in general are set forth in Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla., which is incorporated herein by reference. Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, for example, The Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”), all of which are incorporated herein by reference. Also reference is made to the Manual of Industrial Microbiology and Biotechnology. A. Demain and J. Davies Eds. ASM Press. 1999.
In some embodiments, the recombinant cells encompassed by the invention are grown under batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. A variation of the batch system is a fed-batch fermentation which also finds use in the present invention. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is a system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium (e.g., containing the desired end-products) is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in the growth phase where production of end products is enhanced. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.
In some embodiments, fermentations are carried out a temperature within the range of from about 10° C. to about 60° C., from about 15° C. to about 50° C., from about 20° C. to about 45° C., from about 25° C. to about 45° C., from about 30° C. to about 45° C. and from about 25° C. to about 40° C. Indeed, it is intended that any suitable fermentation temperature will be used in the present invention.
In some other embodiments, the fermentation is carried out for a period of time within the range of from about 8 hours to 240 hours, from about 8 hours to about 168 hours, from about 16 hours to about 144 hours, from about 16 hours to about 120 hours, or from about 24 hours to about 72 hours. Indeed, it is intended that any suitable fermentation time will find use in the present invention.
In some other embodiments, the fermentation will be carried out at a pH in the range of about 4 to about 8, in the range of about 4.5 to about 7.5, in the range of about 5 to about 7, or in the range of about 5.5 to about 6.5. Indeed, it is intended that any suitable pH range will find use in the present invention.
Carbon sources useful in the aqueous fermentation medium (e.g., broth) in which the recombinant microorganisms are grown are those that can be assimilated by the recombinant host strain. Such carbon sources are available in many forms and include renewable carbon sources, including but not limited to cellulosic and starch feedstock substrates obtained therefrom. Such examples include for example fermentable sugars such as monosaccharides, disaccharides, and short chain oligosaccharides (e.g., glucose, fructose, xylose, galactose, arabinose, maltose, mannose, arabinose, and sucrose, as well as numerous other sugars; it is not intended that the present invention be limited to any particular fermentable sugar). Other carbon sources include, but are not limited to saturated and unsaturated fatty acids, glycerol, lactose, succinate, acetate and mixtures thereof.
In some embodiments, the assimilable carbon source is from cellulosic and/or starch feedstock derived from but not limited to, wood, wood pulp, paper pulp, grain (e.g., corn grain), corn stover, corn fiber, rice, paper and pulp processing waste, woody or herbaceous plants and residue, fruit or vegetable pulp, distillers grain, grasses, rice hulls, wheat straw, cotton, hemp, flax, sisal, corn cobs, sugar cane bagasse, sugar beets, sorghum, barely, barely straw, switch grass, wood chips, municipal solid wastes, aquatic crops, and mixtures thereof.
In some embodiments, the cellulosic feedstock useful as an assimilable carbon source has been derived from a biomass substrate that has been pretreated. The term “biomass” is broadly used herein to encompasses any living or dead biological material that contains a polysaccharide substrate, including but not limited to cellulose, starch, other forms of long-chain carbohydrate polymers, and mixtures of such sources. Examples of biomass include, but are not limited to, wood, wood pulp, paper pulp, corn fiber, corn grain, corn cobs, sugar cane, sugar beet, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice, rice straw, switchgrass, waste paper, paper and pulp processing waste, woody or herbaceous plants, fruit or vegetable pulp, distillers grain, grasses, rice hulls, cotton, hemp, flax, sisal, sugar cane bagasse, sorghum, soy, switchgrass, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, and flowers and any suitable mixtures thereof. In some embodiments, the biomass comprises, but is not limited to cultivated crops (e.g., grasses, including C4 grasses, such as switch grass, cord grass, rye grass, miscanthus, reed canary grass, or any combination thereof), sugar processing residues, for example, but not limited to, bagasse (e.g., sugar cane bagasse, beet pulp [e.g., sugar beet], or a combination thereof), agricultural residues (e.g., soybean stover, corn stover, corn fiber, rice straw, sugar cane straw, rice, rice hulls, barley straw, corn cobs, wheat straw, canola straw, oat straw, oat hulls, corn fiber, hemp, flax, sisal, cotton, or any combination thereof), fruit pulp, vegetable pulp, distillers' grains, forestry biomass (e.g., wood, wood pulp, paper pulp, recycled wood pulp fiber, sawdust, hardwood, such as aspen wood, softwood, or a combination thereof). Furthermore, in some embodiments, the biomass comprises cellulosic waste material and/or forestry waste materials, including but not limited to, paper and pulp processing waste, municipal paper waste, newsprint, cardboard and the like. In some embodiments, biomass comprises one species of fiber, while in some alternative embodiments the biomass comprises a mixture of fibers that originate from different biomasses. In some embodiments, the biomass may also comprise transgenic plants that express ligninase and/or cellulase enzymes (See e.g., US 2008/0104724 A1).
In some embodiments, the biomass substrate is “pretreated,” using methods known in the art, such as chemical pretreatment (e.g., ammonia pretreatment, dilute acid pretreatment, dilute alkali pretreatment, or solvent exposure), physical pretreatment (e.g., steam explosion or irradiation), mechanical pretreatment (e.g., grinding or milling) and biological pretreatment (e.g., application of lignin-solubilizing microorganisms) and combinations thereof, to increase the susceptibility of cellulose to hydrolysis. In some embodiments, the substrate is slurried prior to pretreatment. The following references described various means of pretreatment. Steam explosion performing acid pretreatment of biomass substrates is described in U.S. Pat. No. 4,461,648. Continuous pretreatment using a slurry is described U.S. Pat. No. 7,754,457. Methods of alkali pretreatment is such as Ammonia Freeze Explosion, Ammonia Fiber Explosion or Ammonia Fiber Expansion (“AFEX”) are described in U.S. Pat. Nos. 5,171,592; 5,037,663; 4,600,590; 6,106,888; 4,356,196; 5,939,544; 6,176,176; 5,037,663 and 5,171,592. Alternative methods to AFEX utilizing a dilute ammonia pretreatments are described in WO2009/045651 and US 2007/0031953. Chemical pretreatments with organic solvents are disclosed in U.S. Pat. No. 4,556,430. Other pretreatments methods are disclosed in U.S. Pat. No. 7,465,791, and Weil et al. (1997) Appl. Biochem. Biotechnol., 68(1-2): 21-40 [1997].
9. PRODUCTION OF FATTY ALCOHOLS AND SATURATION LEVELS
In various embodiments, fatty alcohols produced by the methods of the invention are further recovered or isolated. Recovery or isolation of the produced fatty alcohols refers to substantially separating the fatty alcohols from other components of the culture medium or fermentation process. Recovery or isolation may be accomplished by solvent extraction of the aqueous nutrient medium with a suitable water immiscible solvent. Extraction may occur simultaneously with fatty alcohol production and in some embodiments, extraction is continuous. Phase separation followed by solvent removal provides the fatty alcohol which may then be further purified and fractionated using methods and equipment known in the art. In some other aspects of the invention, the secreted fatty alcohols coalesce to form a water immiscible phase that can be directly separated from the aqueous nutrient medium either during the fermentation process or after its completion.
In certain embodiments of the invention, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, of the fatty alcohols produced by the methods described herein are secreted into the culture medium by the recombinant host cells.
In certain embodiments, fatty alcohols are isolated by separating the host cells from the aqueous nutrient medium, for example by centrifugation, resuspension and extraction of the fatty alcohols from the recombinant host cells using an organic solvent or solvent mixture. Suitable protocols for recovering fatty alcohols from recombinant host cells and/or culture medium are known to the skilled artisan. In some embodiments, fatty alcohols may be recovered by first lysing the cells to release the fatty alcohols and then extracting the fatty alcohol from the lysate using conventional means. Reference is also made to Yeast Protocols Handbook, (2009) Clontech Laboratories, Inc. A Takara Bio Company, Mt. View Calif. 94043; PNAS 2003 Vol. 100, 16:9156-9161; and Doan et al., (2009) J. Plant Physiol. 166: 787-796 which discloses methods to isolate and measure fatty alcohols produced in E. coli using FARs from Arabidopsis. Indeed, it is intended that any suitable method will find use in the present invention and it is not intended that the present invention be limited to any particular method(s) for separating host cells from the nutrient medium.
In various embodiments, the compositions produced by the engineered cells and methods described herein comprise both saturated and unsaturated fatty alcohols. In certain embodiments, the unsaturated fatty alcohols are monounsaturated fatty alcohols. In some embodiments, the fatty alcohol compositions comprise both saturated and unsaturated fatty alcohols, and the amount of unsaturated fatty alcohols compared to saturated fatty alcohols in the total fatty alcohol composition is less than about 40%, less than about 35%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the fatty alcohols present in the composition.
In some embodiments, the fatty alcohol compositions comprise both saturated and unsaturated fatty alcohols. In some embodiments, the percentage of saturated fatty alcohols in the fatty alcohol compositions produced by the engineered bacterial cells encompassed by the invention is greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 97%.
In some embodiments, the fatty alcohol compositions produced by the engineered cells and the methods described herein comprise one or more alcohols selected from 1-decanol (C10:0), 1-dodecanol (C12:0), 1-tetradecanol (C14:0), 1-hexadecanol (C16:0), and 1-octadecanol (C18:0).
In some typical embodiments, C10 to C18 fatty alcohols comprise at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight of the total fatty alcohols produced by the recombinant host cells. In some embodiments, C12 to C16 fatty alcohols comprise at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, or at least about 98% by weight of the total fatty alcohols produced by the recombinant host cells. In certain embodiments, C14 to C16 fatty alcohols comprise at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, or at least about 99% by weight of the total produced fatty alcohols. It is understood that a reference to a “Cx fatty alcohol” (e.g., C12) includes both saturated and unsaturated fatty alcohols having “x” carbon atoms.
In some typical embodiments, C10:0 to C18:0 fatty alcohols comprise at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight of the total produced fatty alcohols. In some embodiments, C12:0 to C16:0 fatty alcohols comprise at least about 85%, at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 98% by weight of the total produced fatty alcohols. In certain embodiments, C14:0 to C16:0 fatty alcohols comprise at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, or at least about 99% by weight of the total produced fatty alcohols. The proportions of saturated and unsaturated fatty alcohols produced by the strains may be calculated after quantifying all the fatty alcohol species using any suitable method known in the art (e.g., GC-FID as described in US 2011/0000125SA1). The saturated fraction represents the sum of all C12:0-OH; C14:0-OH; C16:0-OH and C18:0-OH. While the unsaturated fraction is composed of the sum of C12:1-OH: C14:1-OH: C16:1-OH and C18:1-OH.
In some embodiments, the fatty alcohol compositions produced by the engineered cells comprise a % of saturated fatty alcohols that is greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 97%. In some additional embodiments, the fatty alcohol compositions further comprise at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, or at least about 98% C12 to C16 fatty alcohols.
In certain embodiments, the present invention provides a recombinant bacterial cell comprising: a) a heterologous fabZ polynucleotide encoding a FabZ enzyme comprising a sequence having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:8 or SEQ ID NO: 26 and b) a heterologous polynucleotide encoding a FAR polypeptide having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:2, SEQ ID NO: 4; SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 28; and wherein the recombinant bacterial host cell produces a fatty alcohol composition that comprises at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C14:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, or about 90%. In some embodiments, the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C12:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90% and about 95%.
In certain aspects of this embodiment, the amount of saturated fatty alcohols produced by the engineered bacterial cell is at least about 1%, at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% greater than the amount of saturated fatty alcohol produced by a corresponding engineered bacterial cell grown under essentially the same culture conditions. In some embodiments, the engineered bacterial cell is one that produces a heterologous FAR and a heterologous FabZ and/or FabI wherein the corresponding bacterial cell to which the engineered bacterial cell of the invention is compared to does not included a heterologous FabZ and/or FabI and the engineered bacterial cell of the invention produces a fatty alcohol composition that is at least 5%, at least 10%, at least 15%, at least 20% or greater than the corresponding bacterial cell to which is it compared to. In some embodiments, the engineered bacterial cell is one that produces a heterologous FAR; a heterologous FabZ and/or a heterologous FabI; and further expresses or overexpresses a heterologous TE and a FadD wherein the corresponding bacterial cell to which the engineered bacterial cell it is compared to does not included a heterologous FabZ and/or heterologous FabI and the engineered bacterial cell of the invention produces a fatty alcohol composition that is at least 5%, at least 10%, at least 15%, at least 20% or greater than the corresponding bacterial cell to which is it compared to. In some embodiments, the engineered bacterial cell is one that produces a heterologous FAR and a heterologous FabA and/or a heterologous FabI wherein the corresponding bacterial cell to which the engineered bacterial cell of the invention is compared to does not included a heterologous FabA and/or FabI and the engineered bacterial cell of the invention produces a fatty alcohol composition that is at least 5%, at least 10%, at least 15%, at least 20% or greater than the corresponding bacterial cell to which is it compared to. In some embodiments, the engineered bacterial cell is one that produces a heterologous FAR and further has an inactivated FadR gene wherein the corresponding bacterial cell to which the engineered bacterial cell is compared to does not included an inactivated FadR gene and the engineered bacterial cell of the invention produces a fatty alcohol composition that is at least 5%, at least 10%, at least 15% at least 20% or greater than the corresponding bacterial cell to which is it compared to. In some embodiments, the engineered bacterial cell is one that produces a heterologous FAR and further has an inactivated FabF gene wherein the corresponding bacterial cell to which the engineered bacterial cell is compared to does not included an inactivated FabF gene and the engineered bacterial cell of the invention produces a fatty alcohol composition that is at least 5%, at least 10%, at least 15% at least 20% or greater than the corresponding bacterial cell to which is it compared to. In some embodiments, the culturing is carried out a temperature within the range of from about 25° C. to about 45° C.
In certain embodiments, the present invention provides a recombinant bacterial cell comprising: a) a heterologous fabI polynucleotide encoding a FabI enzyme comprising a sequence having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12 and b) a heterologous polynucleotide encoding a FAR polypeptide having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 28. In some embodiments, the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C14:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90% and about 95%. In some embodiments, the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C12:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, 90% and 95%. In certain aspects of this embodiment, the amount of saturated fatty alcohols produced by the engineered bacterial cell is at least about 1%, at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% greater than the amount of saturated fatty alcohol produced by a corresponding engineered bacterial cell grown under essentially the same culture conditions. In some embodiments, the culturing is carried out a temperature within the range of from about 25° C. to about 45° C.
In certain embodiments, the present invention provides a recombinant bacterial cell comprising: a) a heterologous fabZ polynucleotide encoding a FabZ enzyme comprising a sequence having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:8 or SEQ ID NO: 26; b) a heterologous fabI1 polynucleotide encoding a FabI1 enzyme comprising a sequence having at least about 80%, about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:12; and/or c) a heterologous polynucleotide encoding a FAR polypeptide having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 28. In some embodiments, the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C14:0 to C16:0 fatty alcohols. In some embodiments, the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C12:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90% and about 95%. In some embodiments, the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, 90% and 95%. In certain aspects of this embodiment, the amount of saturated fatty alcohols produced by the engineered bacterial cell is at least about 1%, at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% greater than the amount of saturated fatty alcohol produced by a corresponding engineered bacterial cell grown under essentially the same culture conditions. In some embodiments, the culturing is carried out a temperature within the range of from about 25° C. to about 45° C.
In certain embodiments, the present invention provides a recombinant bacterial cell comprising: a) a heterologous fabA polynucleotide encoding a FabA enzyme comprising a sequence having at least about 80% about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:10; and b) a polynucleotide encoding a FAR polypeptide having at least about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, about 99%, or even about 100% sequence identity to SEQ ID NO:2; SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 18 or SEQ ID NO: 28, wherein the recombinant bacterial cell produces a fatty alcohol composition that comprises at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C14:0 to C16:0 fatty alcohols, wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, or about 90% or wherein the recombinant bacterial cell produces a fatty alcohol composition comprising at least about 70%, about 75%, about 80%, about 82%, about 84%, about 86%, about 88%, or about 90% C12:0 to C16:0 fatty alcohols wherein the % saturation of the fatty alcohol composition is greater than about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, 90% and 95%. In certain aspects of this embodiment, the amount of saturated fatty alcohols produced by the engineered bacterial cell is at least about 1%, at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% greater than the amount of saturated fatty alcohol produced by a corresponding engineered bacterial cell grown under essentially the same culture conditions. In some embodiments, the culturing is carried out a temperature within the range of from about 25° C. to about 45° C.
In certain embodiments, the amount of fatty alcohols produced by the methods described herein comprise saturated and/or unsaturated C8 to C18 alcohols produced by a recombinant host cell comprising a heterologous gene encoding a FAR as described herein in a range of about 10 mg/L to about 50 g/L of aqueous nutrient medium, such as in a range of about 10 mg/L to about 5 g/L, or in a range of about 10 mg/L to about 2 g/L of medium, using routine modification of culturing conditions. In some embodiments, the amount of fatty alcohols produced by the methods described herein is at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2.0 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, or at least about 10 g/L of medium. In various embodiments, the amount of fatty alcohols produced by the methods described herein is at least about 20 g/L, at least about 30 g/L, at least about 40 g/L, or at least about 50 g/L of medium. In some embodiments, a recombinant bacteria (e.g., E. coli) encompassed by the invention produces C12 to C16 fatty alcohols in an amount of at least about 1.0 g/L, at least about 5.0 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, or at least about 30 g/L of medium. In some embodiments, a recombinant bacteria (e.g., E. coli) encompassed by the invention produces C12 to C14 fatty alcohols in an amount of at least about 1.0 g/L, at least about 5.0 g/L, at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, or at least about 30 g/L of medium. One method to extract and quantify fatty alcohols is provided in US Patent Application 2011/0000125. However, it is not intended that the present invention be limited to any particular method(s) for extracting and/or quantifying the fatty alcohols produced using the present invention, as any suitable methods find use.
In some embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 100 mg/g to about 5 g/g of dry cell weight. In other embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 1 g/g to about 4 g/g of dry cell weight, such as in the range of about 2 g/g to about 3 g/g of dry cell weight.
In certain embodiments, the amount of fatty alcohols produced by the methods described herein is in the range of about 10% to about 20% of dry cell weight, about 20% to about 30% of dry cell weight, about 30% to about 40% of dry cell weight, about 40% to about 50% of dry cell weight, about 50% to about 60% of dry cell weight, about 60% to about 70% of dry cell weight, or about 70% to about 80% of dry cell weight.
In some embodiments, the fatty alcohol compositions produced by the engineered cells and methods described herein may also comprise fatty acid-derived compounds. Fatty acid-derivative compounds include compounds such as but not limited to esters (e.g. acetyl, methyl or ethyl esters and waxes) and fatty acids.
10. COMPOSITIONS
In yet another aspect, the present invention relates to the use of the engineered organisms as described herein for the production of various compositions, including but not limited to, fuel compositions (e.g., biodiesels and petrodiesels), cleaning compositions including detergent compositions (e.g., laundry detergents in liquid gel, spray, and powder form, hard surface cleaners, dishwashing detergents, and the like); industrial compositions (e.g., lubricants, solvents, and industrial cleaners); and personal care compositions (e.g., soaps, cosmetics, shampoos, gels, etc.).
Detergent Compositions
In some embodiments, the fatty alcohol compositions described herein, and compounds derived therefrom, can be used as components of detergent compositions. Detergent compositions comprising fatty alcohols and fatty alcohol derivatives produced by the methods of the present invention include compositions used in cleaning applications, including, but not limited to, laundry detergents, hand-washing agents, dishwashing detergents, rinse-aid detergents, household detergents, and household cleaners, in liquid, gel, granular, powder, or tablet form. In some embodiments, the fatty alcohols produced by the methods described above are used directly in detergent compositions. In some embodiments, the fatty alcohols and fatty alcohol derivatives are reacted with a sulfonic acid group to produce sulfate derivatives that can be used as components of detergent compositions. Detergent compositions that can be generated using the fatty alcohols and fatty alcohol derivatives produced by the methods of the present invention include, but are not limited to, hair shampoos, rinses, and conditioners for humans and other animals, carpet shampoos, hard surface cleaners, light-duty household cleaners, light-duty household detergents, heavy-duty household cleaners, and heavy-duty household detergents. Detergent compositions generally include, in addition to fatty alcohols and derivative thereof, one or more builders (e.g., sodium carbonate, complexation agents, soap, and zeolites), enzymes (e.g., proteases, lipases, cellulases, and/or amylases); carboxymethyl cellulose, optical brighteners, fabric softeners, colourants and perfumes (e.g., cyclohexyl salicylate). Indeed, it is not intended that the present invention be limited to any particular detergent, detergent formulation, nor detergent use.
In some embodiments, sulfate derivatives (e.g., C12-15) derived from fatty alcohols are used in products such as hair shampoos, carpet shampoos, light-duty household cleaners, and light-duty household detergents. In some embodiments, sulfate derivatives (e.g., C16-C18) derived from fatty alcohols are used in products such as hair shampoos and conditioners. In some embodiments, sulfate derivatives (e.g., C16-18) derived from fatty alcohols are used in products such as heavy-duty household cleaners and heavy-duty household detergents. Indeed, it is not intended that the present invention be limited to any particular detergent, detergent formulation, nor detergent use.
Personal Care Compositions
In some embodiments, fatty alcohol compositions as described herein, and compounds derived therefrom, are used as components in personal care compositions. In some embodiments, the fatty alcohols produced by the methods described above are used directly in personal care compositions. Personal care compositions containing fatty alcohols or fatty alcohol derivatives produced by the methods of the present invention include compositions used for application to the body (e.g., for application to the skin, hair, nails, or oral cavity) for the purposes of grooming, cleaning, beautifying, or caring for the body, including but not limited to lotions, balms, creams, gels, serums, cleansers, toners, masks, sunscreens, soaps, shampoos, conditioners, body washes, styling aids, and cosmetic compositions (e.g., makeup in liquid, cream, solid, anhydrous, or pencil form). In some embodiments, the fatty alcohols or fatty alcohol derivatives can be reacted with a sulfonic acid group to produce sulfate derivatives that can be used as components of said compositions. In some embodiments, sulfate derivatives (e.g., C12-14) derived from the fatty alcohol compositions produced by the methods described herein are used in products such as toothpastes. Indeed, it is not intended that the present invention be limited to any particular formulation, nor use.
In some embodiments, fatty alcohol compositions (e.g., C12) produced by the methods described herein are used in products such as lubricating oils, pharmaceuticals, and as an emollient in cosmetics. In some embodiments, fatty alcohol compositions (e.g., C14) produced by the methods described herein are used in products such as cosmetics (e.g., cold creams) for its emollient properties. In some embodiments, fatty alcohol compositions (e.g., C16) produced by the methods described herein are used in products such as cosmetics (e.g., skin creams and lotions) as an emollient, emulsifier, or thickening agent. In some embodiments, fatty alcohol compositions (e.g., C18) produced by the methods described herein are used in products such as lubricants, resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, or thickening agent. Indeed, it is not intended that the present invention be limited to any particular formulation, nor use.
Other Compositions:
In some embodiments, fatty alcohol compositions (e.g., C12) produced by the methods described herein are used in products such as lubricating oils, pharmaceuticals, and as an emollient in cosmetics. In some embodiments, fatty alcohol compositions (e.g., C14) produced by the methods described herein are used in products such as cosmetics (e.g., cold creams) for its emollient properties. In some embodiments, fatty alcohol compositions (e.g., C16) produced by the methods described herein are used in products such as cosmetics (e.g., skin creams and lotions) as an emollient, emulsifier, or thickening agent. In some embodiments, fatty alcohol compositions (e.g., C18) produced by the methods described herein are used in products such as lubricants, resins, perfumes, and cosmetics, e.g., as an emollient, emulsifier, or thickening agent. In some embodiments, sulfate derivatives (e.g., C12-14) derived from the fatty alcohol compositions produced by the methods described herein are used in products such as toothpastes.
In some instances, fatty alcohols (especially cetyl alcohol, stearyl alcohol and myristyl alcohol) may be used as food additives (e.g., adjuvants and production aids).
Alkane and/or Alkene Compositions
In some embodiments, fatty alcohols produced according to the methods described herein can be reduced to yield alkanes and/or alkenes having the same carbon chain length as the fatty alcohol starting materials. Without being bound by any particular theory, the hydroxyl group of an alcohol is a poor leaving group, and therefore, in principle a chemical moiety that binds to the oxygen atom of the hydroxyl group to make it a better leaving group can be used to reduce the fatty alcohols described herein.
Any suitable method known in the art can be used to reduce the fatty alcohols. In some embodiments, reduction of fatty alcohols is carried out chemically, for example, by a Barton deoxygenation (or Barton-McCombie deoxygenation), a two-step reaction in which the alcohol is first converted to a methyl xanthate or thioimidazoyl carbamate, and the xanthate or thioimidazoyl carbamate is reduced with a tin hydride or trialkylsilane reagent under radical conditions to produce the alkane and/or alkene. See Li et al., 2007, Modern Organic Synthesis in the Laboratory, p. 81-83. In another embodiment, alkanes are produced by hydrogenation of fatty alcohols.
The alkanes can be isolated from the reaction mixture (which may contain unreduced fatty alcohols) to yield a composition comprising substantially all alkanes Alternatively, the alkanes and un-reduced fatty alcohols can be isolated from the reaction mixture to yield a composition comprising alkanes and fatty alcohols. In some embodiments, the fatty alcohol compositions comprise at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% alkanes by weight of the composition after reduction. In some embodiments, the alkane is octane, decane, dodecane, tetradecane, hexadecane, octadecane, icosane, docosane, tetracosane, or mixtures thereof.
Ester Compositions:
In other embodiments, fatty alcohols are reacted with a carboxylic acid to form acid esters. Esterification reactions of fatty alcohols are well-known in the art. In certain embodiments, the transesterification reaction is carried out in the presence of a strong catalyst, e.g., a strong alkaline such as sodium hydroxide. In other embodiments, the esterification reaction is carried out enzymatically, using an enzyme that catalyzes the conversion of fatty alcohols to acid esters, such as lipoprotein lipase. See, e.g., Tsujita et al., 1999, “Fatty Acid Alcohol Ester-Synthesizing Activity of Lipoprotein Lipase” J. Biochem. 126:1074-1079.
The following examples are offered to illustrate, but not to limit, the claimed invention.
EXAMPLES
The present invention is described in further detail in the following Examples, which are not in any way intended to limit the scope of the invention as claimed. In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and μg (micrograms); L and l (liter); ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); ° C. (degrees Centigrade); wt % (weight percent); w.r.t. (with regard to); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); gDNA (genomic DNA); cDNA (complementary DNA); Sigma (Sigma Aldrich, St. Louis, Mo.); Qiagen (Qiagen, Inc., Valencia, Calif.); Invitrogen (Invitrogen, Corp., Carlsbad, Calif.); and Promega (Promega, Corp., Madison, Wis.).
Example 1
Construction of Plasmid pLS8379
To overproduce the FAR enzyme having SEQ ID NO:2 in E. coli, a low copy vector carrying the strong Trc promoter was constructed. A DNA fragment containing the lacIq gene, the Trc promoter, and the multiple cloning sites present in pTrcHis2-B (Invitrogen, Carlsbad, Calif.) was PCR amplified using the following primers:







1920TrcM-F:


(SEQ ID NO: 30)


5′ GACCTTAAAACCCTAAAGGCTTAAGGGCATCCGCTTACAGACA 


and


 


1920TrcM-R:


(SEQ ID NO: 31)


5′GGAGAAAATACCGCATCAGGCGCCTCAGGAGAGCGTTCACCGAC.






The PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 65° C. for 15 sec and 72° C. for 15 sec. This was followed by a final elongation step at 72° C. for 5 min.
The primers used for this PCR reaction carry regions of homology to plasmid pCL1920. Because of this, the PCR product described above can be used as a megaprimer to amplify a defined region of pCL1920 (Lerner and Inouye (1990) NAR 18: 4631) which contains the pSC101 origin of replication and confers resistance to Spectinomycin (GenBank: AB236930). This PCR reaction was carried out using the Pfu Ultra enzyme (Agilent Technologies, Santa Clara, Calif.) with an initial denaturation step at 95° C. for 2 min, followed by 16 cycles of the steps: 95° C. for 30 sec; 55° C. for 30 sec and 68° C. for 7 min. This was followed by a final elongation step at 68° C. for 7 min.
The outcome of the second PCR reaction was sequence-verified and the resulting plasmid was named pLS8379 (SEQ ID NO: 29).
Example 2
Construction of Plasmids pCL3079 and Plasmid pCL5019
Two synthetic genes (SEQ ID NO: 3 and SEQ ID NO: 5) encoding the FAR polypeptides having SEQ ID NO: 4 (V1) and SEQ ID NO: 6 (V2) were ligated as NcoI-SalI fragments to pLS8379 digested with the same restriction enzymes. Ligation reactions were incubated overnight at 16° C. and then transformed into E. coli DH10B-T1 electrocompetent cells (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocols. Cells were plated on LB agar plates containing 100 ug/ml of Spectinomycin. Plates were incubated overnight at 37° C. Obtained clones were sequence verified.
Example 3
Construction of the Plasmids pCL3079-fabA; pCL3079-RBSfabA; pCL5019-fabA and pCL3079-RBSfabA
The E. coli fabA gene (SEQ ID NO: 9) (encoding FabA comprising SEQ ID NO:10) including its natural ribosome binding site (RBS) sequence was PCR amplified from genomic DNA isolated from E. coli strain W3110, using primers:









ampFabA-Fwd:



(SEQ ID NO: 32)



5′-TACAGAGAACATGGTAGATAAAC-3′



and



 



ampFabA-Rev:



(SEQ ID NO: 33)



5′-TAGAAGGCAGACGTATCCTG-3′.






Primer ampFabA-Rev was designed to replace the normal TGA stop codon of fabA with the TAA stop codon.
The PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 58° C. for 20 sec and 72° C. for 15 sec. This was followed by a final elongation step at 72° C. for 5 min.
The PCR product obtained was cloned using the TOPO kit from Invitrogen (Carlsbad, Calif.) following manufacturer instructions. Topo cloning reaction was transformed into commercial electrocompetent TOP10 E. coli cells and cells were plated on LB agar plates containing 100 micrograms/ml of Carbenicillin. Plates were incubated overnight at 37° C. The sequence of the cloned fabA gene was verified.
A variant of the fabA gene, where its native ribosome binding site (RBS)/spacing was replaced with a strong RBS/spacing with the sequence: AGGAAACAGCTATG (SEQ ID NO: 34), was obtained by PCR using primer RBS-fabA with the new RBS sequence incorporated on it:









RBS-fabA:



(SEQ ID NO: 35)



5′-AGGAAACAGCTATGGTAGATAAACGCGAATC-3′.






This PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 58° C. for 20 sec and 72° C. for 15 sec. This was followed by a final elongation step at 72° C. for 5 min.
pCL3079-fabA and pCL5019-FabA were constructed as follow:
pCL3079 and pCL5019 were linearized by digestion with the PmeI restriction enzyme. To facilitate cloning, the ends of the DNA fragments were dephosphorylated using Antartic phosphatase (New England BioLabs, Ipswich, Mass.) following manufacturer recommendations.
The two different versions of the fabA genes were PCR amplified from the clones described above using primers containing 20 bp homology to the vector on forward and reverse primer described below:







Pme-inF-RBS-FabA:


(SEQ ID NO: 36)


TCATCATCATCATTGAGTTTAGGAAACAGCTATGGTAGATAAAC;


 


Pme-inF-FabA_Fwd:


(SEQ ID NO: 37)


TCATCATCATCATTGAGTTTTACAGAGAACATGGTAGATAAAC;


and


 


Pme-inF-FabA-Rev:


(SEQ ID NO: 38)


AGCCAAGCTGGAGACCGTTTTTAGAAGGCAGACGTATCCTG.






This PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 58° C. for 20 sec and 72° C. for 15 sec. This was followed by a final elongation step at 72° C. for 5 min.
Obtained PCR products were gel purified using NucleoSpin Extract II Kit (Clontech Laboratories Inc. Mountain View, Calif.) and cloned into linearized pCL3079 or pCL5019 using the In-Fusion Kit (Clontech Laboratories Inc. Mountain View, Calif.) according to manufacturer's protocol. In-Fusion reaction mixture was transformed into DH10B-T1 cells plated on LB agar plates containing 100 micrograms/ml of Spectinomycin. Plates were incubated overnight at 37° C. Resulting plasmids were sequence-verified and named pCL3079-fabA, pCL3079-RBS-fabA, pCL5019-fabA or pCL5019-RBS-fabA respectively.
Example 4
Construction of the pCL3079-fabZ and pCL5019 fabZ
The E. coli fabZ gene (SEQ ID NO: 7) including its natural RBS/spacing sequence was PCR amplified from genomic DNA isolated from E. coli strain W3110, using primers:









ampFabZ-Fwd:



(SEQ ID NO: 39)



5′-ACAGGAAGAGTATCATGACTACTAAC-3′



and



 



ampFabZ-Rev: 



(SEQ ID NO: 40)



5′-TTAGGCCTCCCGGCTACGAGCAC-3′.






The PCR reaction was carried out as described above in Example 3. These primers were also used to replace the original TTG start and TGA stop codons present in fabZ, with ATG and TAA codons respectively. The obtained PCR product was cloned using the TOPO cloning kit as described in Example 3. The sequence of fabZ gene was verified.
The fabZ gene was cloned downstream of FAR in plasmid pCL3079 and pCL5019 using the same approach described above for fabA. The sequence of the primers used to amplify fabZ for the In-Fusion reactions were:









Pme-inF-FabZ-Fwd:



(SEQ ID NO: 41)



ATCATCATCATCATTGAGTTTAGGAAGAGTATCATGACTAC 



and



 



Pme-inF-FabZ-Rev:



(SEQ ID NO: 42)



CAGCCAAGCTGGAGACCGTTTTTAGGCCTCCCGGCTACGAG.






Example 5
Construction of pCL5019-fabI1-fabZ
pCL5019 comprising the polynucleotide encoding the FAR variant having the amino acid sequence of SEQ ID NO:6 (FAR-V2) was digested with restriction enzymes SalI-PmeI (New England BioLabs, Ipswich, Mass.) following the manufacturer recommendations. A synthetic linker containing a multi-cloning site (MCS) was ligated into this vector to facilitate further cloning. pCLlinker_Fwd: TCGACATAGATCTAGAACTTACTCGGAAGCTTCTTAATTAAGAGG ATCCATTGACGTCTATGAATTCGTTT (SEQ ID NO:43) and pCLlinker_Rev: AAACGAATTCATAGACGTCAATGGATCCTCTTAATTAAGAAGCTT CCGAGTAAGTTCTAGATCTATG (SEQ ID NO:44). After ligation, the obtained plasmid pCL5019 linker was sequence verified.
The protein sequence of FabI-1 from Rhodobacter sphaeroides SB103 (GenBank: ADE86080. SEQ ID NO: 12) was utilized to design an E. coli codon-optimized gene (SEQ ID NO: 11). This gene was synthesized and cloned in pUC57 by GenScript (Piscataway, N.J.). The gene was PCR amplified from plasmid pUC57-FabI-1, using Phusion polymerase, with primers containing a SalI site and RBS/spacing in front of fabI-1 and a HindIII site on the 3′ end primer.
Primers Sequences:









5019-FabI-1Fwd:



(SEQ ID NO: 45)



5′-ACTAAGTCGACATAAGGAGATATACATATGACC 



 



5019-FabI-1Rev:



(SEQ ID NO: 46)



5′-AGGTCAAGCTTATTAGTCTTTACG 






The PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 10 sec, followed by 25 cycles of the steps: 98° C. for 20 sec; 56° C. for 20 sec and 72° C. for 30 sec. This was followed by a final elongation step at 72° C. for 5 min.
After the PCR reaction, the PCR product was purified through a PCR purification column and eluted with water. This DNA was digested with the restriction enzymes SalI and HindIII (New England BioLabs, Ipswich, Mass.) following manufacturer recommendations.
The fabZ gene was PCR amplified from TOPO-fabZ plasmid described in Example 5 with primers containing HindIII site on 5′end primer and AatII site on 3′end primer.









FabZ_Fwd:



(SEQ ID NO: 47)



AGTAAGCTTGAGTTTAGGAAGAGTATCATG 



and



 



FabZ_Rev:



(SEQ ID NO: 48)



AAGCTGACGTCTTAGGCCTCCCGGCTACG 






The resulting PCR product was purified through a PCR purification column; eluted with water. The DNA was digested with the restriction enzymes HindIII and AatII (New England BioLabs, Ipswich, Mass.) following manufacturer recommendations.
The final plasmid pCL5019-FabI-1-fabZ was obtained by a three way ligation between FabI-1/SalI-HindII, FabZ/HindIII-AatII fragments and pCL5019 linker vector digested with SalI-AatII. The ligation reaction was performed overnight at 16° C.
The product of the ligation reaction was transformed into E. coli DH10B-T1 electrocompetent cells and plated on LB agar plates containing 100 micrograms/ml of Spectinomycin. Plates were incubated overnight at 37° C. The obtained construct was sequence verified.
Example 6
Construction of pCL5019-FabI1-FabA and pCL5019-FabI1-RBSFabA
A plasmid which overexpresses FAR, the FabI-1 from R. sphaeroides SB103 and the E. coli fabA or RBS-fabA was constructed as described above in Example 5, except that the following primers were used to PCR-amplify fabA:









FabA_Fwd:



(SEQ ID NO: 49)



AGTAAGCTTGAGTTTTACAGAGAACATGG



and



 



FabA_Rev:



(SEQ ID NO: 50)



AAGCTGACGTCTTAGAAGGCAGACGTATCC 



or for



 



RBS-fabA:



rbsFabA_Fwd:



(SEQ ID NO: 51)



AGTAAGCTTGAGTTTAGGAAACAGCTATG 



and 



 



FabA_Rev:



(SEQ ID NO: 52)



AAGCTGACGTCTTAGAAGGCAGACGTATCC 






Example 7
Construction of FadR Deletion (ΔFadR) Strains
Strain JW1176-1 in which the fadR gene (SEQ ID NO:15 and SEQ ID NO: 16) was deleted and replaced by a KmR cassette was obtained from the Coli Genetic Stock Center (Yale University, New Haven, Conn.). This strain is part of the Keio collection constructed by Baba et al., 2006. Mol Syst Biol 2:1-11.
To generate a PCR product containing large regions of homology flanking the fadR::kan region the following primers were used:









FadR-Fwd1:



(SEQ ID NO: 53)



5′-GCCATCGCCAGAGTGAAAATAAATTCCG 



and



 



FadR-Rev1:



(SEQ ID NO: 54)



5′-GATTTCAAAGATGAGAGTTTTATCAGCCAGTTCCT.






This PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 34 cycles of the steps: 98° C. for 10 sec; 56° C. for 20 sec and 72° C. for 1 min. This was followed by a final elongation step at 72° C. for 5 min.
After PCR, the resulting DNA fragment was gel-purified using a QIAquick Gel extraction kit (Qiagen Inc. Valencia, Calif.) following the manufacturer recommended protocol.
Strain W3110-fhuA was transformed with plasmid pKD46 (Coli Genetic Stock Center. Yale University, New Haven, Conn.) and cells were induced with arabinose and electrocompetent cells were prepared as described by Datsenko and Wanner (2000. PNAS 97: 6640-6645). The PCR product obtained above was transformed into the competent cells by electroporation. After 3 hr recovery in SOC media, plated on LB agar plates containing 30 micrograms/ml of Kanamycin. Plates were incubated overnight at 37° C. Resulting colonies were analyzed by colony-PCR using the following verification primers:









FadR-Fwd2:



(SEQ ID NO: 55)



5' TTGGAGCGAATGCTTAACAGCAAACGGG



and



 



FadR-Rev2:



(SEQ ID NO: 56)



5' TGCGCCACTGCTGGAATCATGGCAGCGT.






A colony where the original fadR gene was replaced by the KmR cassette was purified and used for subsequent experiments. This strain was named W3110 fhuA, fadR::kan.
Example 8
Deletion of fabF in Strain W3110-fhuA
A derivative of strain W3110-ΔfhuA was constructed with a deletion of the fabF gene (SEQ ID NO:13) and this strain was named W3110fhuA, fabF::kan. The method used was the same as described above with the following differences:
Strain JW1081-4 (Coli Genetic Stock Center. Yale University, New Haven, Conn.) was used as the source of the fabF::kan mutation. Primers to generate the PCR product were: FabF-U3: 5′ GAAAATTATCGGCGAACAGC (SEQ ID NO:57) and FabF-D3: 5′ GTTAATTAAGAACATACCGGCTCCTTAT (SEQ ID NO:58). Primers to verify the replacement of fabF with the KmR cassette were: FabF-U2: 5′ CTTACATCACGGGTGAAACT (SEQ ID NO:59) and
FabF-D2: 5′ ATCTGCCTGCAACGACTCTT (SEQ ID NO:60).
Example 9
Construction of Deleted fabF (ΔfabF) and fadR (ΔfadR)
Construction of an E. coli strain containing both □fabF and fadR deletions were accomplished by removing the KanR resistance marker present in the W3110ΔfhuA □ΔfadR::kan strain described above in Example 8. Plasmid pCP20 (Coli Genetic Stock Center. Yale University, New Haven, Conn.) is transformed into the strain. The kanamycin marker is removed following the protocol described by Datsenko and Wanner (2000. PNAS 97: 6640-6645). Following this protocol, the KanR is eliminated and a scar with the FRT sequence is left in the chromosome (Datsenko and Wanner, 2000 PNAS 97: 6640-6645)) and (Baba et al., 2006 Mol Sys Biol 2:1-11) resulting in a Kans strain. The fabF gene is then deleted following the procedure described in Example 8. The genotype of this strain is W3110fhuA, fadR::FRT, fabF::kan.
Example 10
Production of Saturated Fatty Alcohols in E. coli Strains Described Above
The proportions of saturated and unsaturated fatty alcohols (“FOHs”) produced by the strains described above were calculated after quantifying all the fatty alcohol species using GC-FID as described in US 2011/0000125SA1.







TABLE 1







% Fatty Alcohol Saturation with Over-expressed fabA.










E. coli strain (W3110 ΔfhuA) +
% FOH saturation



indicated plasmid (p)
in the FOH composition














pCL3079
62.5



pCL3079-fabA
66.7



pCL3079-RBS-fabA
68.6



pCL5019
57.9



pCL5019-fabA
62



pCL5019-RBS-fabA
63.6

















TABLE 2







% Fatty Alcohol Saturation with Over-expressed fabZ.










E. coli strain (W3110 ΔfhuA) +
% FOH saturation



indicated plasmid (p)
in the FOH composition







pCL3079
62.5



pCL3079-fabZ
87.2



pCL5019
57.9



pCL5019-fabZ
84.0

















TABLE 3







% Fatty Alcohol Saturation with Over-expressed fabI1.










E. coli strain (W3110 ΔfhuA) +
% FOH saturation



indicated plasmid (p)
in the FOH composition







pCL5019
58.5



pCL5019-fabI1
52.5



pCL5019-fabI1-fabA
61.0



pCL5019-fabI1-RBSfabA
65.4



pCL5019-fabI1-fabZ
91.6

















TABLE 4







% Fatty Alcohol Saturation in a strain


with deleted fabF::Km mutation










E. coli strain
% FOH saturation



(W3110ΔfhuA)
in the FOH composition







pCL5019
57.5



ΔfabF::Km/pCL5019
67.7

















TABLE 5







% Fatty Alcohol Saturation in a strain with inactivated (Δ) fadR










E. coli strain
% FOH saturation



(W3110ΔfhuA)
in the FOH composition







pCL5019
57.6



pCL5019-ΔfadR
69.8










Example 11
Construction of Plasmid pCK110900-FabI1-fabZ
FabI-1-fabZ genes were PCR amplified from pCL-5019-fabI-1-fabZ described in Example 5 above, with the following primers: 488: 5′-GATACGACCCGTAAACTTGCAACCATTTTTGGC-3′ (SEQ ID NO: 61) and 8379_R CGCTTCTGCGTTCTGATTT-3′ (SEQ ID NO: 62). The PCR reaction was carried out using the enzyme Phusion (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 56° C. for 20 sec and 72° C. for 40 sec. This was followed by a final elongation step at 72° C. for 5 min. The obtained PCR product was purified using a NucleoSpin Gel and PCR clean-up Kit (Clontech Laboratories Inc. Santa Clara Calif.), digested with NdeI and EcoRI enzymes, gel purified and ligated with pCK110900-I-Bla (described in U.S. Pat. No. 7,790,432) and digested with the same restriction endonucleases. The ligation reaction was incubated ON at 16° C. and then transformed into E. coli DH10B-T1 strain. 1 ml of SOC media was added to the transformed cells and cells were allowed to recover for 1 h at 37° C. with shaking at 250 rpm. Cells were plated on LB agar plates containing 30 microgram ml of chloramphenicol. Plates were then incubated overnight at 37° C. The presence of insert was verified by colony PCR with GoTaq GreenMix (Promega Corporation Madison, Wis.) following manufacturer recommendations. The obtained clone was sequence verified. The resulting plasmid was named pCK110900-fabI1-fabZ.
Example 12
Construction of pCDX11
The PTRC promoter present in pLS8379 described in Example 1, was replaced with a synthetic DNA fragment containing a PTRC variant where a symmetrical Lac operator [Sadler et al., 1983, PNAS. 80: 6785-6789] was introduced upstream of the −35 region of PTRC. This promoter was synthesized as an EcoRV-NcoI DNA fragment (GeneScript, Piscataway, N.J.) (SEQ ID NO: 63) and used to replace the EcoRV-NcoI region from pLS8379 previously cut with the same restriction enzymes.









SEQ ID NO: 63:



GATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCA



 



TGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGC



 



CTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCT



 



CAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCA



 



CTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACC



 



GCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCA



 



CGACAGGTTTCCCGACTGGAAAGCGGGCAGTAATAATTTAAA



 



TTGGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGC



 



TTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTG



 



CAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCA



 



CTCCCGTTCTGGATAATGTTTTTTGCGCCGACATAATTGTGA



 



GCGCTCACAATTTCTGAAATGAGCTGTTGACAATTAATCATC



 



CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTC



 



ACACAGGAAACAGCGCCGCTGAGAAAAAGCGAAGCGGCACTG



 



CTCTTTAACAATTTATCAGACAATCTGTGTGGGCACTCGACC



 



GGAATTATCGATTAACTTTATTATTAAAAATTAAAGGAGGAA



 



TAAACCATGG






A ligation reaction containing the two DNA fragments was incubated overnight at 16° C. and then transformed into E. coli Top10 electrocompetent cells (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocols. Cells were plated on LB agar plates containing 100 ug/ml of Spectinomycin. Plates were incubated overnight at 37° C. Obtained clones were sequence verified. The resulting plasmid was named pCDX11.
Example 13
Chromosomal Integration fabI1 and fabZ
(1) The entire lacI structural gene and lacZ promoter were deleted from E. coli strain W3110K using lambda RED-mediated homologous recombination. Cells were transformed with plasmid pSIM5 (Gene. 2006 Sep. 1; 379:109-15). A single colony was grown to log-phase at 32° C., induced at 42° C. for 15 minutes and electrocompetent cells were prepared as described by Datta, Costantino, and Court (2006) Gene 379: 109-115. Competent cells were transformed with 500 ng of the synthetic oligonucleotide lac_del_F: 5′-G*G*A*A*GGCGAAGCGGCATGCATTTACGTTGACACCATCGAATT CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC-3′ (SEQ ID NO: 102), wherein *=phosphorothioate bond modifications incorporated into the synthetic oligonucletide (IDT Technologies, Coralville, Iowa).
After transformation, cells were recovered at 32° C. for five hours, plated on LB agar plates supplemented with 50 ug/ml of X-Gal (Promega, Madison, Wis.), and incubated overnight at 37° C. A white colony from the plate was sequence verified to have a deletion of the lad gene and lacZ promoter and was named strain W3110K-lac-del. Next a double-stranded DNA cassette for inserting the lacIq and pCDX11 sequences into the strain above was generated in two steps:
(a) A dsDNA cassette was PCR amplified from the plasmid pCDX11-5019 described in Example 2, using the following primers: lac-intro_F: 5′-CGCAGGCTATTCTGGTGGCCGGAAGGCGAAGCG GCATGCATTTA-3′ (SEQ ID NO: 64) and lac-intro_R2: 5′-AACGACGGCCAGTGAATCCGTAA TCATGGTCATGGTTTATTCCTCCTTTAATTTTTAATA-3′ (SEQ ID NO: 65). The PCR reaction was carried out using Phusion DNA polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 30 cycles of the steps: 98° C. for 5 sec; 63° C. for 20 sec and 72° C. for 30 sec. A final elongation step was conducted at 72° C. for 5 min. After the PCR reaction, the PCR products were purified through a purification column and eluted with water.
(b) Homology to lacZ was added to the cassette described in part (a) using the following primers: lac-intro_F: 5′-CGCAGGCTATTCTGGTGGCCGGAAGGCGAAGCGGCATGCATTTA-3′ (SEQ ID NO: 66) and lac-intro_R1: 5′-AGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAC GA CGGCCAGTGAATCC-3′ (SEQ ID NO:67). The second PCR was carried out with the same conditions as in part (a). After the PCR reaction, the PCR product was purified through a PCR purification column and eluted with water. This product was named lacIq-PCDX11 cassette.
Strain W3110K-lac-del (see above) was transformed with the plasmid pSIM5. Recombination proficient cells were generated as described above, and the cells were transformed with 500 ng of the lacIq-pCDX11 cassette. After recovery at 32° C. for five hours, the cells were plated on M9 minimal media agar (DifcoTM M9 Minimal Salts, Cat. No. 248510) supplemented with MgSO4, CaCl2, and 0.5% lactose. After several days, lactose proficient colonies emerged on the minimal media plates. One colony was purified by streaking to single colonies and a clone was confirmed to have the lacIq-pCDX11 insertion by sequence verification. The purified clone was named W3110K-lacIq-pCDX11-lacZ.
(2) A fabI-1-fabZ-cat cassette was obtained by PCR using the plasmid pCK110900-fabI-1-fabZ plasmid described in Example 11 above. In this plasmid, the chloramphenicol resistance marker (cat) is located downstream of the fabI1-fabZ genes. The PCR reaction was carried out using the primers and conditions listed below. Nucleotides in italics are homologous to targeted sequences in genomic DNA, while those in underline are homologous to amplification of cassettes.







124.ptrc60.fabI-1.F 


(SEQ ID NO: 68)


5′-GGGCACTCGACCGGAATTATCGATTAACTTTATTATTAAAAATTA


 


AAGGAGGAATAAACCATGACCGCAGGACTGATGGC 


and


 


125.Y550-CAT.R


(SEQ ID NO: 69)


3′-CAAACTGATTATTGATGGTGAACATGATGCCGACAATCGAGG


 


CACACAGCGCCCAGAAAGGACAAGTTTTGGTGA 






The PCR reaction was carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. The final extension step was conducted at 72° C. for 5 min. After PCR, the resulting DNA fragment was digested with DpnI and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol. Strain W3110ΔhuA-lacIq-CDX11p-lacZ described in above was transformed with plasmid pKD46 (E. coli Genetic Stock Center. Yale University, New Haven, Conn.) and cells were induced with arabinose as described by Datsenko and Wanner (PNAS 97: 6640-6645. 2000). Electrocompetent cells were prepared by repeated pelleting and resuspension in cold sterile dH2O at least twice, concentrated 20-fold into 50 ul. The PCR product obtained above was electroporated with a BioRad GenePulser (BioRad, Hercules, Calif.) using a 1 mm gap cuvette at 1.8 kV, 25 μF and 200Ω. Electroporated cells were immediately added to 1 ml SOC and incubated for 2 hours at 37° C. prior to plating on LB agar plates containing 30 μg/ml chlroamphenicol. Colonies were streaked to isolate single colonies. Insertion of the fabI-1-fabZ cassette was verified by colony PCR and by sequencing the PCR product obtained with the following primer set: 138. lacY lacIq.ver.F1 5′-GGCTGGGATCAGGAGGAGAAG-3′ (SEQ ID NO:70) and 137. lacY949.R 5′-AATGATCAGTGGCGCAAAGAACATA-3′ (SEQ ID NO: 71). The colony confirmed to have the fabI1 and fabZ genes inserted was named W3110ΔfhuA::pCDX11-fabI1-fabZ-cat.
Example 14
Generating W3110K-pCDX11-fabI-1-fabZ-cat-frt
A further version of the fabI1-fabZ-cat cassette was constructed. The cat gene was looped out using the Flp-recombinase as described by Datsenko and Wanner (PNAS 97: 6640-6645, (2000).
Construction and integration was as follows:
The fabI-1-fabZ operon under the pCDX11 promoter was amplified from the genomic DNA of W3110ΔfhuA::pCDX11-fabI1-fabZ-cat using the following primers: 156. Proms-F1 5′-CCAGC GTGGACCGCTTGCTGCAAC-3′ (SEQ ID NO: 72) and 170. fabZtm99 R 5′-CCCGGAGTGATCTTATTTC-3′ (SEQ ID NO: 73). The chloramphenicol resistant cassette (cat-frt) was amplified from pKD32 plasmid (Coli Genetic Stock Center. Yale University, New Haven, Conn.) using the following primers: 171. Z-cat.frt.F 5′-GAAATAAGATCACTCCGGGATTCCGGGGATCCGT CGACC-3′ (SEQ ID NO: 103 and 172.cat.frt-Y551.R 5′-CAAACTGATTATTGATGGTGA ACATGATGCCGACAATCGAGGCACACAGCGCCCAGTGTAGGCTGGAGCTGCTTC-3′ (SEQ ID NO: 104).
The PCR reactions were carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. A final extension step was conducted at 72° C. for 5 min. The PCR amplified fragments were digested with DpnI, and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol, and then combined using SOE PCR (Splicing by Overlap Extension PCR, Warrens et al., Gene 1997, 186(1):29-35) standard protocol to obtain full-length of cdx11p-fabI-1-fabZ-cat-frt cassette. The PCR product was purified using gel-extraction using a Zymoclean Gel DNA recovery Kit.
In the final step, the cassette was inserted in the chromosome of 2 different strains; W3110K (E. coli Stock Center strain #7167, New Haven, Conn.) and W3110ΔfhuA by first transforming plasmid pSIM6 (Datta, Costantino, and Court (2006) Gene 379: 109-115). A purified colony from this transformation was used to prepare competent cells by growing the cells to log-phase at 30° C. reaching OD600 of 0.6 and then induced at 42° C. for 15 minutes. Cells were immediately chilled on ice and electrocompetent cells were prepared by repeated pelleting and resuspension in cold sterile dH2O at least twice, concentrated 20-fold into 50 ul. The PCR cassette obtained above was electroporated with a BioRad GenePulser (BioRad, Hercules, Calif.) using a 1 mm gap cuvette at 1.8 kV, 25 μF and 200Ω. Electroporated cells were immediately added to 1 ml SOC and incubated for 2 hours at 37° C. prior to plating on LB agar plates containing 30 μg/ml chlroamphenicol. Colonies were streaked to isolate single colonies, and the insertion of PCDX11-fabI1-fabZ-cat-frt cassette was verified by sequencing the PCR product obtained with the following primer sets: 138. lacY lacIq.ver.F1 GGCTGGGATCAGGA GGAGAAG (SEQ ID NO: 74); 143. FabI-1.Rev TCAGCAGGCTACCACCGTTCG (SEQ ID NO: 75) and 141. FabI-1Fwd TGCGGAACTGGCGTTTTCTTACC (SEQ ID NO: 76) and 183.C3. Rev GTAG AAACTGCCGGAAATCG (SEQ ID NO:77).
The colony confirmed to have the PCDX11-fabI1-fabZ-cat-frt insertion was named W3110K-PCDX11-fabI1-fabZ-cat-frt and W3110ΔfhuA-PCDX11-fabI1-fabZ-cat-frt
Example 15
Chromosomal Integration of fabI1 and fabZ Genes
To express the fabI1 and fabZ constitutively in the chromosome of the E. coli W3110K strain (E. coli Stock Center strain #7167, New Haven, Conn.), a synthetic promoter (see below) was used. These 2 genes were amplified by PCR using 2 primers with the following relevant properties: Primer oJN-1.-F TCTTTCATACAATGACATATTAAAATATCAGCAAGAATTCCAAAGGGTTTTTTTAGGCC TTTGACAGCTAGCTCAGTCCTAGGTATACTGCTAGCATACTAGAGGCCAGCCTGGCCATA AGGAGATATACAT (SEQ ID NO: 78) Nucleotides 1-44 provide a region of homology with the E. coli pseudogene yhiS-2. Nucleotides 45-107 encode a synthetic promoter sequence and the first 12 bases of the mRNA. Nucleotides 108-132 provide a region of homology with plasmid pCK110900-fabI-1-fabZ (see example 11 above) and allow the amplification of the 5′-end of the fabI1-fabZ genes. Primer pCK-chlor_R. TTGAAATACTTCGAATTGATATTCAGACATTTCTGCCCATGTTTGCTGAAA GGACAAGTTTTGGTGACTG (SEQ ID NO: 79). Nucleotides 1-45 provide a region of homology with the E. coli pseudogene yhiS-2. Nucleotides 46-70 provide a region of homology with plasmid pCK110900-fabI-1-fabZ (see Example 11 above) and allow the amplification of the 3′-end of the cat gene present in the plasmid. PCR reaction composition: H2O (33.3 uL); 5× herculase II reaction buffer (10 uL); dNTPmix (10 mM) (1 uL); DNA template 1 uL); FP (1.25 uL); RP (1.25 uL); Herculase II Phusion (0.8 uL); DSMO (1.5 uL) for total volume of 50 uL. PCR Reaction conditions:
















Segment
# of Cycles
Temperature
Duration






















1
1
95
2
min



2
10
95
20
sec





68
20
sec





68
2
min



3
20
95
20
sec





62
20
sec





68
2
min



4
1
68
8
min










After PCR, the resulting DNA fragment was digested with DpnI and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol. The PCR product was used as an integration cassette. For this purpose, strain W3110K was first transformed with plasmid pSIM6 and competent cells were prepared as described by Datta et al., [Gene 379: 109-115 (2006)]. Briefly, cells were grown to log-phase at 30° C. reaching OD600 of 0.6 and then induced at 42° C. for 15 minutes. Cells were then immediately chilled on ice and electrocompetent cells were prepared by repeated pelleting and resuspension in cold sterile dH2O at least twice. After the final wash, cells were concentrated 20-fold by resuspending them in the appropriate dH2O volume. 50 μl aliquots of this concentrate were used for electroporation. The PCR cassette obtained above was electroporated with a BioRad GenePulser (BioRad, Hercules, Calif.) using a 1 mm gap cuvette at 1.8 kV, 25 μF and 200Ω. Electroporated cells were immediately added to 1 ml SOC and incubated for 2 hours at 37° C. prior to plating on LB agar plates containing 30 μg/ml chlroamphenicol. Colonies were streaked to isolate single colonies. The insertion of Pconst-fabI-1-fabZ-cat cassette was verified by colony PCR and by sequencing the PCR product obtained with the following primer set: ins-yhiS_seqF: ACCAGCCTTGGGGTAAAACG (SEQ ID NO: 80) and ins-yhiS_seqR: TTTTGGGTTAAGC GTCTCGT (SEQ ID NO: 81). The strain confirmed to have Pconst-fabI1-fabZ gene insertion was named W3110K-Pconst-fabI1-fabZ-cat.
Example 16
Construction of pCDX11-8087-fabI-1-RcfabZ
To determine the effect of overexpressing the fabZ gene from Rhodobacter capsulatus on fatty alcohol production in E. coli which also included a polynucleotide encoding the FAR-V3 (SEQ ID NO: 28), plasmid pCDX11-8087-fabI1-RcfabZ was constructed as described below.
A. The cloning vector pCDX11-8087-MCS was linearized by digestion with SalI and Bsu36I restriction enzymes (New England BioLabs, Ipswich, Mass.), and then gel extracted using Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol.
B. The fabI1 gene was amplified from plasmid pCDX11-8087-fabI1-fabZ using the following primers: 237.N-link-fabI-1.F
CTGACGATAAAACCGCCTAAGTCGACATAAGGAGATATACAT (SEQ ID NO: 82) and 238.C-link-fabI-1.R CATGATACTCTTCCTAAACTCAAGCTTATTAGTC (SEQ ID NO: 83).
C. The protein sequence of the fabZ gene from Rhodobacter capsulatus (GenBank:ADE85379, also SEQ ID NO: 26) was utilized to design an E. coli codon optimized gene. To facilitate further manipulations of the gene, other sequences were included upstream and downstream of the structural fabZ gene. The extra downstream sequences comprised a multicloning site (MCS), a transcriptional terminator sequence, and 15 bp of homologous sequence to pCDX11-8087. The extra sequence upstream of the gene contained a region of homology to fabI1. The sequence of the synthetic DNA is illustrated as SEQ ID NO:25 and was synthesized by GenScript (Piscataway, N.J.), who provided it cloned into pUC57 and named pUC57-RcfabZ. The fabZ gene was amplified from plasmid pUC57-RcfabZ using the following primers: 239.I-1-RcZ.F TCGGCATGAAAGCGGTTGATG (SEQ ID NO: 84) and 241.Bsu36I.21.R GAGAAAATACCGCATCAGGCG (SEQ ID NO: 85).
The PCR reactions was carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. This was followed by a final extension step at 72° C. for 5 min. The two PCR amplified fragments described in sections B and C above, were digested with DpnI, and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit, and then combined using SOE PCR (Splicing by Overlap Extension PCR, Warrens et al., Gene 1997, 186(1):29-35) standard protocol using the primers 237.N-link-fabI-1.F and 241.Bsu36I.21.R shown above. These primers contain 20 bp of homologous sequence to the plasmid pCDX11-8087. The PCR product was purified using gel-extraction using a Zymoclean Gel DNA recovery Kit and cloned into linearized pCDX11-8087 described in section A above, using the In-Fusion Kit (Clontech Laboratories Inc. Mountain View, Calif.) according to manufacturer's protocol. In-Fusion reaction mixture was transformed into DH10B-T1 (Invitrogen, CA) cells using electroporation method described in Example 15. Transformed cells were plated on LB agar agar containing 100 μg/ml of spectinomycin. Plates were incubated overnight at 37° C. Resulting plasmid was sequence-verified and named pCDX11-8087-fabI1-RcfabZ.
Example 17
Construction of pCDX11-8087-fabI1-fabZ
Plasmid pCDX11-8087-FabI-1-FabZ overexpressing the fabI1-fabZ genes as part of an operon with the gene encoding the FAR-V3 under control of the CDX11 promoter was constructed as described below.
First, the E. coli codon-optimized FabI-1 gene from Rhodobacter sphaeroides SB103 and the native E. coli fabZ gene were PCR amplified from plasmid pCL-5019-FabI-1-FabZ (see Example 5), using primers 488_F and 8379_R: 488_F GATACGACCCGTAAACTTGCAACCATTTTTGGC (SEQ ID NO: 86) and 8379_R CGCTTCTGCGTTCTGATTT (SEQ ID NO: 87).
The PCR reaction was carried out using Phusion DNA polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 30 cycles of the steps: 98° C. for 5 sec; 63° C. for 20 sec and 72° C. for 45 sec. A final elongation step was conducted at 72° C. for 5 min. The PCR products were purified through a PCR purification column and eluted with water.
The FabI-1-FabZ PCR product and the plasmid pCDX11-8087-MCS were digested with the restriction enzymes SalI and AatII (Fermentas, Glen Burnie, Md.) and the resulting products were ligated using Quick T4 DNA Ligase (New England BioLabs, Ipswich, Mass.) following manufacturer recommendations. The reaction was transformed into E. coli Top10 electrocompetent cells (Invitrogen, Carlsbad, Calif.) following the manufacturer's protocols. Cells were plated on LB agar plates containing 100 micrograms/ml of spectinomycin and were incubated overnight at 37° C. Obtained clones were sequence verified. This plasmid was named pCDX11-8087-fabI1-fabZ.
Example 19
Construction of pCDX11-8087-fabI1
Plasmid pCDX11-8087-fabI1 was constructed by subcloning the fabI1 gene downstream of the FAR V3 present in plasmid pCDX11-8087-MCS. Plasmid pCDX11-8087-MCS was linearized by digestion with SalI and Bsu36I restriction enzymes (New England BioLabs, Ipswich, Mass.), and then gel extracted using Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol.
The fabI1 gene was PCR amplified from pCDX11-8087-fabI1-fabZ described in example 18, using the following primers: 249.fabI-1.F CTGACGATAAAACCGCCTAAGTCGACAT AAGGAGATATACATATGACCG (SEQ ID NO: 88) and 250. fabI-1-3HA.R AAACGAATTCATAGACGTCAAGCTTATTAGTCTTTACGACCCGTGAC (SEQ ID NO: 89).
For the In-Fusion reaction, the downstream sequence of fabI1 requires a homology region to the cloning vector pCDX11-8087-MCS. The nucleotide sequence for the homology region of the fabI1 gene was obtained from the downstream sequence of RcfabZ present in plasmid pUC57-RcfabZ (see, Example 16 above) and modified using the following primers: 247.I-1-HAEP.F CACGGGTCGTAAAGACT AATAAGCTTGACGTCTATGAATTCGTTTAAACGCCAGGC (SEQ ID NO: 90) and 241.Bsu36I.21.R GAGAAAATACCGCATCAGGCG (SEQ ID NO: 91).
The PCR reaction was carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. This was followed by a final extension step at 72° C. for 5 min. These two PCR amplified fragments were digested with DpnI, and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit, and then combined using SOE PCR (Splicing by Overlap Extension PCR, Warrens et al., Gene 1997, 186(1):29-35) standard protocol using the primers 249.fabI-1.fw and 241.Bsu36I.21.R shown above. These primers contain 20 bp of homologous sequence to the plasmid pCDX11-8087. The PCR product was purified using gel-extraction using a Zymoclean Gel DNA recovery Kit and cloned into linearized pCDX11-8087-MCS (described above) using the In-Fusion Kit (Clontech Laboratories Inc. Mountain View, Calif.) according to manufacturer's protocol. In-Fusion reaction mixture was transformed into DH10B-T1 (Invitrogen, CA) cells using electroporation method shown in example 2. Transformed cells were plated on LB agar agar containing 100 μg/ml of spectinomycin. Plates were incubated overnight at 37° C. The resulting plasmid was sequence-verified and named pCDX11-8087-fabI1.
Example 18
Construction of pCDX11-8087-RcfabZ
The cloning vector pCDX11-8087-MCS was linearized by digestion with SalI and Bsu36I restriction enzymes (New England BioLabs, Ipswich, Mass.), and then gel extracted using Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, Calif.) following the manufacturer recommended protocol. The RcfabZ gene was amplified from plasmid pUC57-RcfabZ described in Example 16 using the following primers containing 20 bp of homologous sequence to the cloning vector pCDX11-8087: 248. Far-RcZ.fw GACGATAAAACCGCCTAATAAGCTTGAGTTTAGGAAGAGTATC ATGTCTG (SEQ ID NO:92) and 241.Bsu36I.21.R GAGAAAATACCGCATCAGGCG (SEQ ID NO: 93).
This PCR reaction was carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. This was followed by a final elongation step at 72° C. for 5 min. After PCR, the resulting DNA fragment was digested with DpnI and purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit. The RcfabZ gene fragment was cloned into linearized pCDX11-8087-MCS using the In-Fusion Kit (Clontech Laboratories Inc. Mountain View, Calif.) according to manufacturer's protocol. In-Fusion reaction mixture was transformed into DH10B-T1 (Invitrogen, CA) cells using electroporation method shown in example 2. Transformed cells were plated on LB agar agar containing 100 μg/ml of spectinomycin. Plates were incubated overnight at 37° C. Resulting plasmid was sequence-verified and named pCDX11-8087-RcfabZ.
Example 19
Construction of pCDX11-8087-MCS Plasmid
The plasmid pCDX11-FAR-MCS comprising the polynucleotide encoding FAR-V3 was constructed as follows: A DNA fragment containing the FAR-V3 gene was PCR amplified using the primers: 8087_NcoI_F 5′-TAAACCATGGCGACTCAACAACAGAACA (SEQ ID NO: 94) and 8087_SalI_R 5′-CTATGTCGACTTAGGCGGTTTTATCGTCAGTATCA (SEQ ID NO: 95). The PCR reaction was carried out using the Phusion polymerase (New England BioLabs, Ipswich, Mass.) with an initial denaturation step at 98° C. for 30 sec, followed by 25 cycles of the steps: 98° C. for 10 sec; 60° C. for 20 sec and 72° C. for 15 sec/kb. This was followed by a final extension step at 72° C. for 5 min. After PCR, the resulting DNA fragment was purified by gel-extraction using a Zymoclean Gel DNA Recovery Kit. As the restriction enzyme sites NcoI and SalI were incorporated into the primers 8087_NcoI_F and 8087_SalI_R respectively, this allowed the ligation of this PCR product into pCDX11 digested with NcoI and SalI restriction enzymes accordingly the manufacturer conditions (New England BioLabs, Ipswich, Mass.). Ligation reactions were incubated overnight at 16° C. and then transformed into E. coli TOP10 chemically competent cells (Invitrogen, Carlsbad, Calif.) using standard techniques. Cells were plated on LB agar plates containing 100 ug/ml of Spectinomycin and incubated overnight at 37° C. Obtained clones were sequence verified. A clone with the correct sequence was designated pCDX11-8087-MCS.
Example 20
Construction of pCDX11-8087-BTE1-fadD
To produce fatty alcohols from acyl-CoA intermediates instead of acyl-ACP intermediates, a plasmid to overexpress FAR, the Bay tree thioesterase (BTE) and the E. coli acyl-CoA synthase gene (FadD) was constructed as follows.
The polynucleotide (SEQ ID NO: 27) encoding variant FAR-V3 (SEQ ID NO: 28) was PCR amplified using pCDX11-8087-MCS using the following primers:









5′ cloning site + RBS1:FAR 8087 



(SEQ ID NO: 96)



5′ CCGGAATTATCGATTAACTTTATTATTAAAAATT



 



AAAGGAGGAATAAACCATGGCGACTCAACAACAGAAC



and



 



FAR 8087:RBS-ptrc



(SEQ ID NO: 97)



3′ TAAGGTCATGGTTTATTCCTCCTTGTCGACTTAGGCG



 



GTTTTATCGTCAGTATC.






The polynucleotide encoding the BTE thioesterase (SEQ ID NO: 21) was synthesized by GenScript (Piscataway, N.J.), and the synthesized gene was amplified by PCR using the following primers:









RBS-ptrc:CaBayTES1



(SEQ ID NO: 98)



5′ ACCGCCTAAGTCGACAAGGAGGAATAAACCATGACCTTA



 



GAGTGGAAACCAAAA



and



 



CaBayTES1:RBS-pCK 



(SEQ ID NO: 99)



3′ GCCAAACCTTCTTCATATGTATATCTCCTTTTATACCCG



 



CGGCTCGG.






The native E. coli fadD gene (SEQ ID NO: 19) was PCR amplified from E. coli genomic DNA using the following primers:









RBS-pCK:fadD 5′:



(SEQ ID NO: 100)



CGAGCCGCGGGTATAAAAGGAGATATACATATGAAGAAGGTT



 



TGGCTTAACCG



and



 



fadD:3′ cloning site 3′: 



(SEQ ID NO: 101)



TTAAGAAGCTTCCGAGTAAGTTCTAGATCTTCATTAGGCTTT



 



ATTGTCCACTTTG.






PCR amplifications were performed with Herculase II (Agilent Technologies, Santa Clara, Calif.) following manufacturer's protocol with at 60° C. annealing temp. These three PCR amplified fragments were combined using SOE PCR (splicing by overlap extension PCR) standard protocol (see, Warrens et al., 1997 Gene 186(1):29) using primers 5′ cloning site+RBS1:FAR 8087 5′ and fadD:3′ cloning site 3′ mentioned above. The final PCR product was inserted into linearized pCDX11 plasmid with ClaI and BglII restriction enzymes from Fermentas (Thermo Scientific, Glen Burnie, Md.), ligated overnight at 16° C. with T4 DNA ligase per manufacturer's protocol (NEB, Ipswich, Mass.), and transformed into electrocompetent W3110 ΔfhuA using standard molecular biology methods (Dower et al., 1988 NAR 16:6127-6145). Cells were plated on LB agar plates containing 100 ug/ml of spectinomycin and plates were incubated overnight at 37° C. Clones were sequence-verified and the plasmid named pCDX11-8087-BTE-fadD.
Example 21
Fatty Alcohol Production from E. coli Strains Described Above
The amount of fatty alcohols and saturation level by the strains described above were calculated after quantifying all the fatty alcohols species using GC_ED as described in US2011/0000125SA1.







TABLE 6







Fatty alcohol levels and % saturation produced in E. coli


W3110ΔfhuA strains with fabI1-fabZ overexpressed with


an inducible promoter integrated in the chromosome.











Plasmid
g/L FOH
% Saturation







pCDX11-5019
2.35
63.5



pCDX11-fabI1-fabZ-cat/
2.77
76.7



pCDX5019

















TABLE 7







Fatty alcohol levels and % saturation produced in E. coli


W3110ΔfhuA strains with fabI1-fabZ overexpressed with


an inducible promoter integrated in the chromosome.











Plasmids
g/L FOH
% Saturation















pCDX115019-BTE1-fadD
1.4
67.4



pCDX117076-BTE1-fadD
3.33
82.5



pCDX11-fabI1-fabZ-cat-frt/
3.18
66.7



pCDX5019-BTE1-fadD



pCDX11-fabI1-fabZ-cat-frt/
4.58
83.5



pCDX7076-BTE1-fadD










    
    
        Plasmid pCDX11-7076-BTE1-fadD was constructed in the same manner as
        pCDX11-5019 and pCDX11-8086-BTE1-fadD, but with a FAR variant having 2 amino acid residues different than FAR-V3.
    
    









TABLE 8







Fatty alcohol levels and % saturation produced in E. coli


W3110K strains with fabI1-fabZ overexpressed with a plasmid.











Plasmids
g/L FOH
% Saturation















pCDX11-8087-BTE-fadD
3.8
53.5



pCDX11-fabI1-fabZ-cat-frt/
4.25
70.0



pCDX8087-BTE-fadD

















TABLE 9







Fatty alcohol levels and % saturation produced in E. coli


W3110K strains with fabI1-fabZ overexpressed with a constitutive


promoter integrated in the chromosome.











Plasmids
g/L FOH
% Saturation







pCDX11-8087-BTE1-fadD
3.8
53.5



Pconst-fabI1-fabZ-cat/
3.6
67.7



pCDX8087-BTE1-fadD

















TABLE 10







% saturated fatty alcohols in E. coli W3110 ΔfhuA strains overexpressing


the Rhodobacter capsulatus fabI1 and/or fabZ genes.










Plasmids
% Saturation







pCDX11-8087-fabI1
59



pCDX11-8087-RcFabZ
88



pCDX11-8087-fabI1-FabZ
82



pCDX11-8087-fabI1-RcFabZ
90










All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

## Claims

1. An engineered bacterial microorganism comprising at least one polynucleotide encoding at least one heterologous fatty acyl-ACP reductase enzyme (FAR), wherein said FAR comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:28 and has fatty alcohol forming acyl-CoA reductase activity, and a first recombinant polynucleotide encoding a FabZ enzyme, wherein said FabZ enzyme comprises the amino acid sequence set forth in SEQ ID NO:8, wherein the engineered bacterial microorganism produces a fatty alcohol composition comprising C10 to C18 fatty alcohols.

2. The engineered bacterial microorganism according to claim 1 further comprising a second recombinant polynucleotide encoding a FabI enzyme.

3. The engineered bacterial microorganism of claim 2, wherein the FabI enzyme comprises an amino acid sequence comprising at least 80% sequence identity to SEQ ID NO:12, and has enoyl ACP-reductase activity.

4. An engineered bacterial microorganism comprising a polynucleotide encoding i) a heterologous fatty acyl-ACP reductase enzyme having at least 95% sequence identity to SEQ ID NO: 28 and fatty alcohol forming acyl-CoA reductase activity, ii) a first recombinant polynucleotide sequence encoding a FabZ enzyme having at least 95% sequence identity to SEQ ID NO:8 and dehydratase activity, and iii) a second recombinant polynucleotide sequence encoding a FabI enzyme having at least 95% sequence identity to SEQ ID NO: 12 and enoyl-ACP reductase activity, wherein the engineered bacterial microorganism is capable of producing a fatty alcohol composition.

5. A method of producing a fatty alcohol composition comprising:
a) providing the engineered microorganism of claim 1;
b) culturing the engineered microorganism in a culture medium under suitable culture conditions for the production of fatty alcohols or derivatives thereof;
c) allowing production of the fatty alcohols or derivatives thereof; and
d) optionally recovering the produced fatty alcohols or derivative thereof from the engineered microorganism.

6. The method according to claim 5, wherein the fatty alcohol composition comprises at least 60% of C12:0, C14:0, C16:0 and C18:0 fatty alcohols.

7. The method according to claim 5, wherein the % of saturated fatty alcohols in the fatty alcohol composition is at least 60%.

8. The method according to claim 5, wherein the culturing is at a temperature of about 25° C. to about 45° C.

9. The method according to claim 5, wherein at least 2 g/L of fatty alcohols are produced.

10. A method for producing a fatty alcohol composition comprising at least 60% saturated fatty alcohols comprising:
a) providing the engineered bacterial microorganisms of claim 1;
b) culturing the engineered microorganism in a culture medium under suitable culture conditions for the production of fatty alcohols;
c) allowing production of the fatty alcohols; and
d) optionally recovering the produced fatty alcohols.

11. The method according to claim 10, wherein the fatty alcohol composition comprises at least 60% of saturated C12, C14, C16 and/or C18 fatty alcohols.

12. The method according to claim 10, wherein at least 2 g/L of fatty alcohols are produced.