# Patent

## Title
Surfactant and cleaning compositions comprising microbially produced branched fatty alcohols

## Abstract

The invention provides a surfactant and/or a cleaning composition comprising a microbially produced branched fatty alcohol or a derivative thereof. The invention also provides a household cleaning composition and a personal or pet care cleaning composition comprising a microbially produced branched fatty alcohol or a derivative thereof.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 61/304,448, filed Feb. 14, 2010, and U.S. Provisional Patent Application No. 61/324,310, filed Apr. 15, 2010, the entire contents of which are hereby incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 415,467 bytes ASCII (Text) file named “707643_ST25” created Apr. 11, 2011.


BACKGROUND OF THE INVENTION
Fatty alcohols have many commercial uses. Worldwide annual sales of fatty alcohols and their derivatives are in excess of US$1 billion. Fatty alcohols are used in diverse industries. For example, they are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols can be formulated or be used per se as nonionic surfactants, which are useful in personal care and household products, for example, in detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.
One major use for fatty alcohols is in cleaning compositions. On the other hand, fatty alcohols find applicability as surfactants, which are, for example, capable of enhancing oil recovery and/or engine performance. Conventional surfactants comprise molecules having at least one water-solublizing substituent or moiety (e.g., hydrophilic group) and at least one oleophilic substituent or moiety (e.g., hydrophobic group). Examples of hydrophilic groups include, without limitation, carboxylate, sulfate, sulfonate, amine oxide, or polyoxyethylene. Examples of the hydrophobic groups include, without limitation, alkyl, alkenyl, or alkaryl hydrophobes, which typically contain about 10 to about 20 carbon atoms.
Surfactants are typically regarded as the major force behind cleaning products' ability to break up stains, solubilize dirt and soil, and/or prevent their redeposition to surfaces. As such, surfactants are also referred to as wetting agents and foamers, which lower the surface tension of the medium in which they are dissolved. Capable of lowering the interfacial tension between two media or interfaces (e.g., air/water, water/oil, or oil/solid interfaces), surfactants play a key role, and are often the most important component in detergents. Conventional detergent compositions contain mixtures of various surfactants in order to remove different types of soils and stains from surfaces.
The earliest utilized source of hydrophobe groups were natural fats and oils, which were converted into soaps (e.g., carboxylate hydrophile) using base via saponification processes. Coconut and palm oils are to this day used to manufacture soaps and alkylsulfate surfactants. As edible oils became more scarce, it has become increasingly prevalent to manufacture detergents from petrochemicals, using processes such as the Zeigler process to convert petroleum derived ethylene to fatty alcohols. For example, ethylene has been converted into alkyl benzene sulfonate surfactants, which are commonly found in today's detergents and cleaning compositions.
Fatty alcohols can also served as starting materials in the preparation of surfactants and of other cleaning composition ingredients including, for example, alkyl sulfates, fatty ether sulfates, fatty alcohol sulfates, fatty phosphate esters, alkylbenzyl dimethylammonium salts, fatty amine oxides, alkyl polyglucosides, and alkyl glyceryl ether sulfonates. Among these, alkyl sulfates are commonly known due to the ease of their manufacture as well as their improved solubility and surfactant characteristics over traditional soap-based surfactants. However, long-chain alkyl surfactants have less than optimal performance as surfactants or as component(s) of detergents at low temperatures (e.g., about 50° C. or lower, about 30° C. or lower).
While there have been isolated reports that branching, especially towards the middle part of the long-chain alkyl, can reduce solubility of the surfactant, others have described that, in commercial practices, branching in fatty alcohols is highly desirable. See, e.g., R. G. Laughlin, The Aqueous Phase Behavior of Surfactants,” Academic Press, N.Y., (1994), at page 347; but see, Finger et al., Detergent alcohols—the effect of alcohol structure and molecular weight on surfactant properties, J. Amer. Oil Chemicals Society, Vol. 44:525 (1967); Technical Bulletin, Shell Chemical Co., SC:164-80. In addition, K. R. Wormuth, et al., Langmuir, vol 7 (1991):2048-2053, describes the technical advantages observed with a number of branched alkyl sulfates, especially with the “branched Guerbet” type, derived from the highly branched “Exxal” alcohols (Exxon). Phase studies have established a lipophilic ranking (i.e., a hydrophobicity ranking) if highly branched/double tail>methyl branched>linear. Furthermore, patents and applications, including, for example, U.S. Pat. No. 6,008,181 indicates that certain branched or multi-branched fatty alcohol derivatives exhibit improved cleaning capacity, especially at lower temperatures.
Branched fatty alcohols and various precursors are known to have additional preferred properties such as considerably lower melting points, which can in turn confer lower pour points when made into industrial chemicals, as compared to linear alcohols of comparable molecular weights. They are also known to confer substantially lower volatility and vapor pressure, and improved stability against oxidation and rancidity than their linear counterparts. These additional preferred properties, in addition to making branched materials desirable surfactants, make them particularly suited as components or feedstocks for cosmetic and pharmaceutical applications, as components of plasticizers for making synthetic resins, as solvents for solutions for printing ink and specialty inks, or as industrial lubricants.
Those added preferred properties can be alternatively obtained from unsaturated fatty alcohols and precursors. But unsaturation promotes oxidation, leading to short shelf lives and corrosion. Thus desirable properties, e.g., lower melting points, pour points, volatility, and vapor pressure and improved oxidative stability, are better achieved via branching.
Obtaining branched materials from crude petroleum requires a significant financial investment as well as consumes a great deal of energy. It is also an inefficient process because frequently it is necessary to crack the long chain hydrocarbons in crude petroleum to produce smaller monomers, which only then become useful as raw materials for manufacturing complex specialty chemicals. Furthermore, it is commonplace in the petrochemical industry to obtain branched chemicals, such as branched alcohols and aldehydes, by isomerization of straight-chain hydrocarbons. Expensive catalysts are typically required for isomerization, thus increasing manufacturing cost. The catalysts often then become undesirable contaminants that are removed from the finished products, adding yet further cost to the processes.
Obtaining specialty chemicals such as branched alcohols or derivatives from crude petroleum also drains the dwindling resource of petroleum, in addition to the cost and problems associated with exploring, extracting, transporting, and refining. One estimate of world petroleum consumption is 30 billion barrels per year. By some estimates, it is predicted that at current production levels, the world's petroleum reserves could be depleted before 2050.
Finally, processing and manufacturing of surfactants and/or detergents from petroleum inevitably releases greenhouse gases (e.g., in the form of carbon dioxide) and other forms of air pollution (e.g., carbon monoxide, sulfur dioxide, etc.). The accumulation of greenhouse gases in the atmosphere can lead to increase global warming, causing local pollutions and spillage as well as global environmental detriments.
Thus, although it is possible to obtain branched fatty alcohols and derivatives from natural oils and petroleum, it would be desirable to produce these branched materials from other sources, such as directly from biomass.
SUMMARY OF THE INVENTION
The invention provides a surfactant composition and a cleaning composition comprising one or more microbially produced branched fatty alcohols, branched fatty alcohol precursors, or branched fatty alcohol derivatives thereof.
The invention provides a surfactant composition comprising about 0.001 wt. % to about 100 wt. % of one or more microbially produced branched fatty alcohols or branched alcohol derivatives thereof.
The invention also provides a liquid cleaning composition comprising (a) about 0.1 wt. % to about 50 wt. % of one or more microbially produced branched fatty alcohols or derivatives thereof, or about 0.1 wt. % to about 50 wt. % of a surfactant comprising one or more microbially produced branched fatty alcohols or derivatives thereof, (b) about 1 wt. % to about 30 wt. % of one or more co-surfactants, (c) about 0 wt. % to about 10 wt. % of one or more detergency builders, (d) 0 wt. % to about 2 wt. % of one or more enzymes, (e) about 0 wt. % to about 15 wt. % of one or more chelating agents, (f) about 0 wt. % to about 20 wt. % of one or more hydrotropes, (g), about 0 wt. % to about 1.0 wt. % of one or more organic sequestering agents, and (h) about 0.1 wt. % to about 98 wt. % of a solvent system. In some embodiments, the liquid cleaning composition further comprises one or more suitable adjuncts.
The invention further provides a solid cleaning composition comprising (a) about 0.1 wt. % to about 50 wt. % of one or more microbially produced branched fatty alcohols or derivatives thereof, or about 0.1 wt. % to about 50 wt. % of a surfactant comprising one or more microbially produced branched fatty alcohols or derivatives thereof, (b) about 1 wt. % to about 30 wt. % of one or more co-surfactants, (c) about 1 wt. % to about 60 wt. % of one or more detergency builders, (d) about 0 wt. % to about 2 wt. % of one or more enzymes, (e) about 0 wt. % to about 20 wt. % of one or more hydrotropes, (f) about 10 wt. % to about 35 wt. % of one or more filler salts, (g) about 0 wt. % to about 15 wt. % of one or more chelating agents, and (g) about 0.01 wt. % to about 1 wt. % of one or more organic sequestering agents. In certain embodiments, the solid cleaning composition further comprises one or more suitable adjuncts.
In particular embodiments, the invention pertains to a household cleaning composition comprising (a) about 0.1 wt. % to about 50 wt. % of one or more microbially produced branched fatty alcohols and/or derivatives thereof, or about 0.1 wt. % to about 50 wt. % of a surfactant comprising one or more microbially produced fatty alcohols and/or derivatives thereof; (b) about 1 wt. % to about 30 wt. % of one or more co-surfactants; (c) about 0 wt. % to about 30 wt. % of one or more detergency builders; (d) about 0 wt. % to about 2.0 wt. % of one or more suitable detersive enzymes; (e) about 0 wt. % to about 15 wt. % one or more chelating agents; (f) about 0 wt. % to about 20 wt. % of one or more hydrotropes, (g) about 0 to about 15 wt. % of one or more rheology modifier; (h) about 0 wt. % to about 1.0 wt. % of one or more organic sequestering agents; and (i) various other adjuncts such as, for example, one or more of bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents, and corrosion inhibitors. In an exemplary embodiment, a laundry composition can also comprise softening agents, fragrances, bleach systems, dyes or colorants, preservatives, germicides, fungicides, fabric care benefit agents, gelling agents, antideposition agents, and other detersive adjuncts
Such a household cleaning composition can be a liquid, which further comprises water and/or a suitable aqueous carrier or solvent. Liquid compositions can be in a “concentrated” form, the density of which can range from, for example, about 400 to about 1,200 g/L, when measured at 200° C. For example, the water content of a typical concentrated liquid detergent is less than about 40 wt. %, or less than about 30 wt. %. Alternatively, a household cleaning composition can be a solid, for example, in the form of a tablet, a bar, a powder or a granule. Granular compositions can also be in a “compact” form, which is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of solid cleaning compositions, present in substantial amounts, varying from, for example, about 10 wt. % to about 35 wt. %. Suitable filler salts include, for example, alkali and alkaline-earth metal salts of sulfates and chlorides. An exemplary filler salt is sodium sulfate.
In another embodiment, the invention provides a personal or beauty care cleaning or treatment composition comprising (a) about 0.1 wt. % to about 50 wt. % of one or more microbially produced branched fatty alcohols and/or derivatives thereof, or about 0.1 wt. % to about 50 wt. % of a surfactant comprising one or more microbially produced branched fatty alcohols and/or derivatives thereof; (b) about 0.001 wt. % to about 30 wt. % of one or more co-surfactants; (c) about 0 wt. % to about 30 wt. % of one or more detergency builders; (d) about 0 wt. % to about 2.0 wt. % of one or more suitable detersive enzymes; (e) about 0 wt. % to about 15 wt. % one or more chelating agents; (f) about 0 wt. % to about 20 wt. % of one or more hydrotropes, (g) about 0 to about 15 wt. % of one or more rheology modifier; (h) about 0 wt. % to about 1.0 wt. % of one or more organic sequestering agents; and (i) various other adjuncts such as, for example, one or more of conditioner, silicone, fragrances, silica particles, cationic cellose or guar polymers, silicone microemulsion stabilizers, fatty amphiphiles, germicides, fungicides, anti-dandruff agents, pearlescent agents, foam boosters, pediculocides, pH adjusting agents, UV absorbers, sunscreens, skin active agents, vitamins, minerals, herbal/fruit/food extracts, sphingolipids, sensory indicators, suspension agents, and mixtures thereof.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A and FIG. 1B are schematics of two exemplary alternative pathways for producing branched fatty alcohols using recombinant microbial host cells.
FIG. 2A lists representative homologs of BKD E1 alpha subunit, their amino acid sequences and polynucleotide sequences, as well as amino acid sequence motifs of suitable BKD E1 alpha subunit homologs and variants. FIG. 2B lists representative homologs of BKD E1 beta subunit, their amino acid sequences and polynucleotide sequences, as well as amino acid sequence motifs of suitable BKD E1 beta subunit homologs and variants. FIG. 2C lists representative homologs of BKD E2 subunit, their amino acid sequences and polypeptide sequences, as well as amino acid sequence motifs of suitable BKD E2 subunit homologs and variants. FIG. 2D lists representative homologs of BKD E3 subunit homologs and variants, as well as amino acid sequence motifs of suitable BKD E3 subunit homologs and variants. FIG. 2E lists representative homologs of beta ketoacyl-ACP synthase homologs, their amino acid sequences and polynucleotide sequences, as well as amino acid sequences of suitable beta keto-acyl-ACP synthase homologs and variants.
FIG. 3A is a table of BKD E1 alpha subunit homologs. FIG. 3B is a table of BKD E1 beta subunit homologs. FIG. 3C is a table of BKD E2 subunit homologs. FIG. 3D if a table of BKD E3 subunit homologs. FIG. 3E is a table of beta ketoacyl-ACP synthase homologs. These tables also present % identity in reference to the sequences of various organisms. For example, “ID % Pp” indicates that the identity listed in the column below are in reference to a P. putida gene encoding that subunit. “ID % Bs” refers to the identity to a B. subtilis gene encoding that subunit. “ID % Sc” and “ID % Sc2” refer to identity to a first and second S. coelicolor genes encoding that subunit, respectively. “ID % Sa” and “ID % Sa2” refer to identity to a first and a second S. avermitilis genes encoding that subunit, respectively.
FIG. 4A depicts a GC/MS trace of branched fatty alcohol production of strain MG1655_ΔtonA AAR:kan transformed with a pGL10 vector containing P. putida Pput1450, Pput1451, Pput1452 and Pput1453 inserts, and with B. subtilis fabH1. The figure indicates the production of iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0 and anteiso-C17:0 branched fatty alcohols. FIG. 4B depicts the production of branched fatty acyl-CoA precursors by feeding branched substrates isobutyrate and isovalerate to an engineered E. coli strain comprising the pDG10 and an OP-180 plasmids, the latter plasmid contained teas under the control of a Ptrc promoter.
FIG. 5 is a representative calibration curve obtained by linear regression, which was used in the semi-quantitative measurement of the amount of branched fatty alcohol yield relative to the amount of straight-chain fatty alcohol yield.
FIG. 6A is a listing of nucleotide sequence of the pDG2 plasmid. FIG. 6B depicts a map of the pDG6 plasmid. FIG. 6C is a listing of nucleotide sequence of the pDG6 plasmid, constructed by inserting B. subtilis fabH1 into pDG2, comprising E. coli PfabH1 (promoter) and B. subtilis fabH1. The B. subtilis fabH1 insert is in upper case italic letters. FIG. 6D depicts a map of the pDG7 plasmid. FIG. 6E is a listing of nucleotide sequence of the pDG7 plasmid, constructed by inserting a B. subtilis fabH2 into pDG2, comprising E. coli PfabH1 (promoter) and B. subtilis fabH2. FIG. 6F depicts a map of the pDG8 plasmid. FIG. 6G is a listing of nucleotide sequence of pDG8 plasmid, constructed by inserting S. coelicolor fabH into pDG2, comprising E. coli PfabH1 (promoter) and S. coelicolor fabH. FIG. 6H is a plasmid map of the pDG10 plasmid. FIG. 6I is listing of nucleotide sequence of the pDG10 plasmid, comprising a C. acetobutylicum ptb_buk insert. FIG. 6J is a listing of nucleotide sequence of the pLS9-111 plasmid. FIG. 6K is a listing of nucleotide sequence of the pLS9-114 plasmid. FIG. 6L is a listing of nucleotide sequence of the pLS9-115 plasmid.
FIG. 7 is a listing of nucleotide sequence of the pKZ4 plasmid having a pGL10.173B vector backbone and a polynucleotide insert encoding a BKD complex from Pseudomonas putida. The P. putida genes encoding a BKD complex are shown in lower case italic letters.
FIG. 8 is a listing of nucleotide sequence of the pGL10.173B vector backbone, which contains the BamHI and EcoRI sites to which the Pseudomonas putida bkd genes (operon) were inserted. The BamHI and EcoRI restriction sites are marked.
FIG. 9 is a listing of additional nucleotide and amino acid sequences of the disclosure.



DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein, including GenBank database sequences, are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
DEFINITIONS
Throughout the specification, a reference may be made using an abbreviated gene name or polypeptide name, but it is understood that such an abbreviated gene or polypeptide name represents the genus of genes or polypeptides. Such gene names include all genes encoding the same polypeptide and homologous polypeptides having the same physiological function. Polypeptide names include all polypeptides and homologous polypeptides that have the same activity (e.g., that catalyze the same fundamental chemical reaction).
Unless otherwise indicated, the accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. Unless otherwise indicated, the accession numbers are as provided in the database as of December 2009.
EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (available at www.chem.qmul.ac.uk/iubmb/enzyme/). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of October 2008.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “about” is used herein to mean a value±20% of a given numerical value. Thus, “about 60%” means a value of between 60±(20% of 60) (i.e., between 48 and 70).
The term “alkyl” is used herein to mean a straight chain or a branched chain hydrocarbon residue having from about 6 carbon atoms to about 26 carbon atoms and in the context of the present specification is used interchangeably with the term “fatty.”
As used herein, the term “alcohol dehydrogenase” (EC 1.1.1.*) refers to a polypeptide capable of catalyzing the conversion of a fatty aldehyde to an alcohol (e.g., fatty alcohol). In certain embodiments, these enzymes can also be referred to as fatty aldehyde recutases, oxidoreductases, or aldo-keto reductases. Additionally, one of ordinary skill in the art will appreciate that some alcohol dehydrogenases will catalyze other reactions as well. For example, some alcohol dehydrogenases will accept other substrates in addition to fatty aldehydes. Such non-specific alcohol dehydrogenases are, therefore, also included in this definition. Nucleic acid sequences encoding alcohol dehydrogenases are known in the art, and such alcohol dehydrogenases are publicly available. Exemplary GenBank Accession Numbers are provided in Table 8 herein.
As used herein, the term “attenuate” means to weaken, reduce, or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (e.g., by modifying a nucleotide sequence that encodes the polypeptide) or its expression level.
As used herein, the term “biomass” refers to any biological material from which a carbon source is derived. In some instances, a biomass is processed into a carbon source, which is suitable for bioconversion. In other instances, the biomass may not require further processing into a carbon source. The carbon source can be converted into a fatty alcohol. One exemplary source of biomass is plant matter or vegetation. For example, corn, sugar cane, or switchgrass can be used as biomass. Another non-limiting example of biomass is metabolic wastes, such as animal matter, for example cow manure. In addition, biomass may include algae and other marine plants. Biomass also includes waste products from industry, agriculture, forestry, and households. Examples of such waste products that can be used as biomass are fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers. Biomass also includes carbon sources such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
As used herein, the phrase “carbon source” refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO2). These include, for example, various monosaccharides, such as glucose, fructose, mannose, and galactose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as xylose and arabinose; disaccharides, such as sucrose, maltose, and turanose; cellulosic material, such as methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acid esters, such as succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof. The carbon source can also be a product of photosynthesis, including, but not limited to, glucose. A preferred carbon source is biomass. Another preferred carbon source is glucose.
A nucleotide sequence is “complementary” to another nucleotide sequence if each of the bases of the two sequences matches (i.e., is capable of forming Watson-Crick base pairs). The term “complementary strand” is used herein interchangeably with the term “complement”. The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
As used herein, the term “conditions sufficient to allow expression” means any conditions that allow a host cell to produce a desired product, such as a polypeptide or fatty alcohol described herein. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, such as temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Exemplary culture media include broths or gels. Generally, the medium includes a carbon source, such as glucose, fructose, cellulose, or the like, that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
To determine if conditions are sufficient to allow expression, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as TLC, HPLC, GC/FID, GC/MS, LC/MS, and MS, can be used.
It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated (i.e., will not adversely affect desired biological properties, such as carboxylic acid reductase activity) can be determined as described in Bowie et al., Science, 247: 1306-1310 (1990). A “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
As used herein, “control element” means a transcriptional and/or a translational control element. Control elements include promoters and enhancers, such as ribosome binding sequences. The term “promoter element,” “promoter,” or “promoter sequence” refers to a DNA sequence that functions as a switch that activates the expression of a gene. If the gene is activated, it is said to be transcribed or participating in transcription. Transcription involves the synthesis of mRNA from the gene. A promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Control elements interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237 (1987)).
As used herein, the term “detergent” refers broadly to agents and materials that are useful in cleaning applications or as cleaning aids. This term is thus used interchangeably with the term “cleaning composition.” The term encompasses materials and agents that comprise various surfactants at various percentages by weight or by volume, as well as suitable additives, and are capable of emulsifying stains in a cleaning matrix. A detergent can take the physical form of, for example, a liquid, a paste, a gel, a bar, a powder, a tablet, or a granule. Granular compositions can also be in “compact” form, whereas liquid compositions can be in “concentrate” form.
As used herein, detergent compositions include articles and compositions of cleaning and/or treatment. As used herein, the term “cleaning and/or treatment composition” includes, unless otherwise indicated, tablet, granular, or power-form all-purpose or “heavy duty” washing agents, especially laundry detergents; liquid, gel, or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid types; liquid fine-fabric detergents; hand dishwashing agents, or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablets, granular, liquid and rinse-aid types for household and institutional use. The compositions can also be in unit dose packages, including those known in the art and those that are water soluble, water insoluble and/or water permeable.
As used herein, detergent composition also include personal or beauty care products in the form of skin and hair care compositions including, for example, conditioning treatments, cleansing products, such as hair and/or scalp shampoos, body washes, hand cleaners, water-less hand sanitizers/cleansers, facial cleansers, and the like.
As used herein, the term “fatty acid” means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise about 4 or more carbon atoms. In some embodiments, the fatty acid comprises between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. In addition, fatty acids can comprise a straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches. In a preferred embodiment, the fatty acid is made from a fatty acid biosynthetic pathway.
As used herein, the term “fatty acid biosynthetic pathway” means a biosynthetic pathway that produces fatty acids. The fatty acid biosynthetic pathway includes fatty acid enzymes that can be engineered, as described herein, to produce fatty acids, and in some embodiments can be expressed with additional enzymes to produce fatty acids having desired carbon chain characteristics.
As used herein, the term “fatty acid derivative” means products made in part from the fatty acid biosynthetic pathway of the production host organism. “Fatty acid derivative” also includes products made in part from acyl-ACP or acyl-ACP derivatives. The fatty acid biosynthetic pathway includes fatty acid synthase enzymes which can be engineered as described herein to produce fatty acid derivatives, and in some examples can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics. Exemplary fatty acid derivatives include, for example, fatty acids, acyl-CoA, fatty aldehyde, short and long chain alcohols, hydrocarbons, fatty alcohols, and esters (e.g., waxes, fatty acid esters, or fatty esters), although due to their separate and industrial utilities and depending the sources from which they derive, hydrocarbons can sometimes be grouped into a separate “hydrocarbon” category.
As used herein, the term “fatty acid derivative enzyme” means any enzyme that may be expressed or overexpressed in the production of fatty acid derivatives. These enzymes may be part of the fatty acid biosynthetic pathway. Non-limiting examples of fatty acid derivative enzymes include fatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoA reductases, alcohol dehydrogenases, alcohol acyltransferases, fatty alcohol-forming acyl-CoA reductases, carboxylic acid reductases (e.g., fatty acid reductases), acyl-ACP reductases, fatty acid hydroxylases, acyl-CoA desaturases, acyl-ACP desaturases, acyl-CoA oxidases, acyl-CoA dehydrogenases, ester synthases, and/or alkane biosynthetic polypeptides, etc. Fatty acid derivative enzymes can convert a substrate into a fatty acid derivative. In some examples, the substrate may be a fatty acid derivative that the fatty acid derivative enzyme converts into a different fatty acid derivative.
As used herein, “fatty acid enzyme” means any enzyme involved in fatty acid biosynthesis. Fatty acid enzymes can be expressed or overexpressed in host cells to produce fatty acids. Non-limiting examples of fatty acid enzymes include fatty acid synthases and thioesterases.
As used herein, “fatty aldehyde” means an aldehyde having the formula RCHO characterized by an unsaturated carbonyl group (C═O). In a preferred embodiment, the fatty aldehyde is any aldehyde made from a fatty acid or fatty acid derivative. In one embodiment, the R group is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons in length, or is a value between any two of the foregoing values.
R can be straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches.
Furthermore, R can be saturated or unsaturated. If unsaturated, the R can have one or more points of unsaturation.
In one embodiment, the fatty aldehyde is produced biosynthetically.
Fatty aldehydes have many uses. For example, fatty aldehydes can be used to produce many specialty chemicals. For example, fatty aldehydes are used to produce polymers, resins, dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals. Some are used as solvents, preservatives, or disinfectants. Some natural and synthetic compounds, such as vitamins and hormones, are aldehydes.
The terms “fatty aldehyde biosynthetic polypeptide”, “carboxylic acid reductase”, and “CAR” are used interchangeably herein.
As used herein, “fatty alcohol” means an alcohol having the formula ROH. In a preferred embodiment, the fatty alcohol is any alcohol made from a fatty acid or fatty acid derivative. In one embodiment, the R group is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons in length, or is a value between any two of the foregoing values. Typically, the fatty alcohol comprises an R group that is 6 to 26 carbons in length. Preferably, the fatty alcohol comprises an R group that is 8, 10, 12, 14, 16, or 18 carbons in length.
R can be straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches. In a particular embodiment, the fatty alcohol of the present invention comprises one or more points of branching.
Furthermore, R can be saturated or unsaturated. If unsaturated, the R can have one or more points of unsaturation.
In one embodiment, the branched fatty alcohol is produced biosynthetically.
Fatty alcohols have many uses. For example, fatty alcohols can be used to produce various specialty chemicals. As such, fatty alcohols are used as a biofuel; as solvents for fats, waxes, gums, and resins; in pharmaceutical salves, emollients, and lotions; as lubricating-oil additives; in detergents and emulsifiers; as textile antistatic and finishing agents; as plasticizers; as nonionic surfactants; in cosmetics, e.g., as thickeners.
The term “fatty alcohol derivative” refers to a compound derived from a fatty alcohol. The fatty alcohol derivative can include the oxygen atom derived from the fatty alcohol, or, in some embodiments, does not include the aforesaid oxygen atom, in, for example, fatty amine oxides. For example, a fatty amide, which also can be referred to as an alkyl amide, refers to a compound comprising an amide group and a hydrocarbon residue having about 6 carbon atoms or more, wherein the hydrocarbon residue is bonded to the carbonyl group of the amide group or to the nitrogen atom of the amide group. In some embodiments, the hydrocarbon residue of the fatty alcohol is bonded to the carbonyl group of the amide group or to the nitrogen atom of the amide group. In some embodiments, the hydrocarbon residue is saturated. In other embodiments, the hydrocarbon residue is monounsaturated. In further embodiments, the hydrocarbon residue is polyunsaturated. In certain other embodiments, the hydrocarbon residue can be a straight-chain residue. In certain further embodiments, the hydrocarbon residue can contain one or more points of branching.
Branched fatty alcohols have particularly beneficial properties as compared to their corresponding straight-chain isomers (i.e., isomers of the same molecular weight). For example, branched fatty alcohols tend to have considerably lower melting points when compared to their corresponding straight-chain isomers. Lower melting points confer lower pour points. In addition, branched fatty alcohols tend to substantially lower volatility and vapor pressure, and improved stability against oxidation and rancidity, as compared to their corresponding straight-chain isomers. These beneficial properties render particular suitability of using branched fatty alcohols and/or derivatives thereof as components or feedstocks for cosmetic and pharmaceutical applications, as components of plasticizers for synthetic resins, as solvents for solutions for printing ink and specialty inks, or as industrial lubricants. These materials are also well suited as components of surfactants that have good low-temperature detersive performance. As such, they are especially desirable as ingredients of various household and/or personal care cleaning/treatment compositions wherein low washing temperatures are preferred.
As used herein, “fraction of modern carbon” or “fM” has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1.
“Gene knockout”, as used herein, refers to a procedure by which a gene encoding a target protein is modified or inactivated so as to reduce or eliminate the function of the intact protein. Inactivation of the gene may be performed by general methods such as mutagenesis by UV irradiation or treatment with N-methyl-N′-nitro-N-nitrosoguanidine, site-directed mutagenesis, homologous recombination, insertion-deletion mutagenesis, or “Red-driven integration” (Datsenko et al., Proc. Natl. Acad. Sci. USA, 97: 6640-45 (2000)). For example, in one embodiment, a construct is introduced into a host cell, such that it is possible to select for homologous recombination events in the host cell. One of skill in the art can readily design a knock-out construct including both positive and negative selection genes for efficiently selecting transfected cells that undergo a homologous recombination event with the construct. The alteration in the host cell may be obtained, for example, by replacing through a single or double crossover recombination a wild type DNA sequence by a DNA sequence containing the alteration. For convenient selection of transformants, the alteration may, for example, be a DNA sequence encoding an antibiotic resistance marker or a gene complementing a possible auxotrophy of the host cell. Mutations include, but are not limited to, deletion-insertion mutations. An example of such an alteration includes a gene disruption (i.e., a perturbation of a gene) such that the product that is normally produced from this gene is not produced in a functional form. This could be due to a complete deletion, a deletion and insertion of a selective marker, an insertion of a selective marker, a frameshift mutation, an in-frame deletion, or a point mutation that leads to premature termination. In some instances, the entire mRNA for the gene is absent. In other situations, the amount of mRNA produced varies.
Calculations of “homology” between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent homology between two amino acid sequences is determined using the Needleman and Wunsch (1970), J. Mol. Biol. 48:444 453, algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent homology between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna. CMP matrix and a gap weight of about 40, 50, 60, 70, or 80 and a length weight of about 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Other methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in, for example, Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene 73:237 244, 1988; Higgins & Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; Huang et al., CABIOS 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. and Altschul et al., J. Mol. Biol. 215:403-410, 1990.
As used herein, a “host cell” is a cell used to produce a product described herein (e.g., a branched fatty alcohol described herein). A host cell can be modified to express or overexpress selected genes or to have attenuated expression of selected genes. Non-limiting examples of host cells include plant, animal, human, bacteria, yeast, or filamentous fungi cells.
As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference, and either method can be used. An example of hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions); 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2.×SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions in 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions of 4) are the preferred conditions unless otherwise specified.
The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the nucleic acid. Moreover, by an “isolated nucleic acid” is meant to include nucleic acid fragments, which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, which are isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of chemical precursors or other chemicals when chemically synthesized.
As used herein, the “level of expression of a gene in a cell” refers to the level of mRNA, pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and degradation products encoded by the gene in the cell.
As used herein, the term “microorganism” means prokaryotic and eukaryotic microbial species from the domains Archaea, Bacteria, and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” (i.e., cells from microbes) and “microbes” are used interchangeably and refer to cells or small organisms that can only be seen with the aid of a microscope.
As used herein, the term “nucleic acid” refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides, ESTs, chromosomes, cDNAs, mRNAs, and rRNAs.
As used herein, the term “operably linked” means that selected nucleotide sequence (e.g., encoding a polypeptide described herein) is in proximity to a promoter to allow the promoter to regulate expression of the selected DNA. In addition, the promoter is located upstream of the selected nucleotide sequence in terms of the direction of transcription and translation. By “operably linked” is meant that a nucleotide sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
As used herein, “overexpress” means to express or cause to be expressed a nucleic acid or polypeptide in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell. For example, a polypeptide can be “overexpressed” in a recombinant host cell when the polypeptide is present in a greater concentration in the recombinant host cell compared to its concentration in a non-recombinant host cell of the same species.
As used herein, “partition coefficient” or “P” is defined as the equilibrium concentration of a compound in an organic phase divided by the concentration at equilibrium in an aqueous phase (e.g., fermentation broth). In one embodiment of a bi-phasic system described herein, the organic phase is formed by the fatty aldehyde or fatty alcohol during the production process. However, in some examples, an organic phase can be provided, such as by providing a layer of octane, to facilitate product separation. When describing a two phase system, the partition characteristics of a compound can be described as log P. For example, a compound with a log P of 1 would partition 10:1 to the organic phase:aqueous phase. A compound with a log P of −1 would partition 1:10 to the organic phase:aqueous phase. By choosing an appropriate fermentation broth and organic phase, a branched fatty aldehyde or branched fatty alcohol with a high log P value can separate into the organic phase even at very low concentrations in the fermentation vessel.
As used herein, the term “purify,” “purified,” or “purification” means the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free, preferably at least about 75% free, and more preferably at least about 90% free from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of branched fatty aldehyde or branched fatty alcohol in a sample. For example, when branched fatty alcohols are produced in a host cell, the branched fatty alcohols can be purified by the removal of host cell proteins, or by simply separating and removing linear fatty alcohols that are produced during the same process. After purification, the percentage of branched fatty alcohols in the sample is increased.
The terms “purify,” “purified,” and “purification” do not require absolute purity. They are relative terms. Thus, for example, when branched fatty alcohols are produced in host cells, a purified branched fatty alcohol is one that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other compounds, such as, for example, linear fatty alcohols). In another example, a purified branched fatty alcohol preparation is one in which the branched fatty alcohol is substantially free from contaminants, such as those that might be present following fermentation. In some embodiments, a branched fatty alcohol is purified when at least about 50% by weight of a sample is composed of the branched fatty alcohol. In other embodiments, a branched fatty alcohol is purified when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weight of a sample is composed of the branched fatty alcohol.
As used herein, the term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed protein or RNA is transferred into a suitable expression vector and that is in turn used to transform a host cell to produce the polypeptide or RNA.
As used herein, the term “substantially identical” (or “substantially homologous”) is used to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, such as involving conservative amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.
As used herein, the term “surfactants” refers broadly to surface active agents. These agents are typically amphipathic molecules comprising both hydrophilic and hydrophobic moieties that partition preferentially at the interface between fluid phases with different degrees of polarity and hydrogen bonding, such as, for example, an oil/water interface, or an air/water interface. Surfactants are capable of reducing surface and interfacial tension and forming microemulsions. These characteristics confer detergency, emulsifying, foaming and dispersing traits, making them some of the most versatile process chemicals.
Surfactants can be natural or synthetic in origin. Surfactants from natural origin can be derived from, for example, vegetable or animal sources. Surfactants derived from synthetic origin are typically those derived from petroleum.
There are many types of surfactants, including, for example, anionic surfactants, cationic surfactants, non-ionic surfactants, and amphoteric/zwitterionic surfactants, each with distinct characteristics.
The hydrophobic end of an anionic surfactant is negatively charged in solution. As a result, they have good cleaning properties and high sudding potentials, which make them particularly effective as some of the most widely used types of surfactants in, for example, laundry detergents, dishwashing liquids, and shampoos. Known anionic surfactants include, for example, alkyl sulfates, alkyl ethoxylate sulfates, and soaps.
The hydrophobic end of a cationic surfactant is positively charged in solution. Three types of cationic surfactants are the most commonly known. The first type is the esterquat, which is widely included in, for example, fabric treatment agents or softeners and in detergents with built-in softeners. This is because esterquat is capable of adding softness to fabrics. The second type is a mono alkyl quaternary system, which is found in many household cleaners due to its disinfecting and/or sanitizing properties.
Non-ionic surfactants do not have an electrical charge in solution, making them resistant to water hardness deactivation. They are typically excellent grease removers. The most commonly used non-ionic surfactants are ethers or derivatives of fatty alcohols.
Amphoteric/zwitterionic surfactants are milder than the other types of surfactants, making them particularly suitable for use in personal or beauty care cleaning/treatment products. They may contain two oppositely-charged groups. While the positive charge is typically conferred by ammonium, the source of the negative charge can vary. For example, the negative charge can be conferred by carboxylate, sulfate, sulfonate, or a combination thereof. They can be anionic (e.g., negatively charged), cationic (e.g., positively charged) or non-ionic (e.g., no charge) in solution, depending on the acidity or pH of the solution. They have good compatibility with the other types of surfactants and are well known for being soluble and effective in the presence of high concentrations of electrolytes, acids and alkalis. An example of an amphoteric/zwitterionic surfactant is an alkyl betaine.
In typical applications, different types of surfactants are blended or otherwise used together to achieve an array of desirable properties.
As used herein, the term “synthase” means an enzyme that catalyzes a synthesis process. As used herein, the term synthase includes synthases, synthetases, and ligases.
As used herein, the term “transfection” means the introduction of a nucleic acid (e.g., via an expression vector) into a recipient cell by nucleic acid-mediated gene transfer.
As used herein, “transformation” refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA. This may result in the transformed cell expressing a recombinant form of an RNA or polypeptide. In the case of antisense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.
As used herein, a “transport protein” is a polypeptide that facilitates the movement of one or more compounds in and/or out of a cellular organelle and/or a cell.
As used herein, a “variant” of polypeptide X refers to a polypeptide having the amino acid sequence of peptide X in which one or more amino acid residues is altered. The variant may have conservative changes or nonconservative changes. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
The term “variant,” when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a gene or the coding sequence thereof. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference polynucleotide, but will generally have a greater or fewer number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid sequence identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. In the present specification, “plasmid” and “vector” are used interchangeably, as the plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.
Surfactants and Cleaning Compositions Comprising a Microbially Produced Branched Fatty Alcohol or a Branched Fatty Alcohol Derivative Thereof
The invention provides a surfactant composition comprising one or more microbially produced branched chain fatty alcohols and/or derivatives thereof. The invention further provides a detergent/cleaning composition, such as, for example, a household cleaning composition or a personal or beauty care cleaning composition, comprising such a surfactant.
In one aspect, the invention features a surfactant composition comprising branched chain fatty alcohols and/or derivatives thereof produced by microbes. In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing genes encoding at least one subunit of a branched-chain alpha-keto acid dehydrogenase polypeptide. The host cell expresses genes encoding at least two subunits of a branched-chain alpha-keto acid dehydrogenase polypeptide. For example, the host cell expresses a set of genes encoding the first subunit and a second subunit of a branched-chain alpha-keto acid dehydrogenase polypeptide. In certain embodiments, the host cell expresses a third gene encoding the second subunit of a branched-chain alpha-keto acid dehydrogenase polypeptide. In some embodiments, the first and second polypeptides have branched-chain alpha-keto acid decarboxylase activity, and the third polypeptide has lipoamide acyltransferase activity. In further embodiments, the host cell expresses a fourth gene encoding the third subunit of a branched-chain alpha-keto acid dehydrogenase polypeptide. In some embodiments, the fourth polypeptide has lipoamide dehydrogenase activity.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding a beta ketoacyl-ACP synthase polypeptide. In certain embodiments, the beta ketoacyl-ACP synthase polypeptide has FabH activity. In certain embodiments the beta ketoacyl-ACP synthase has specificity for branched-chain acyl-CoA substrates.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a set of genes encoding at least one subunit of a branched-chain alpha-keto acid dehydrogenase complex. Specifically, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a first gene encoding a first polypeptide comprising the amino acid sequence that is any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15, or one that has 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%, 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% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or a variant thereof; a second gene encoding a second polypeptide comprising an amino acid sequence of any one of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, and 38, or one that has 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%, 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% sequence identity to the amino acid sequence of any one of SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, and 38, or a variant thereof. In certain embodiments, the host cell also expresses a third gene encoding a third polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:47, 49, 51, 53, 55, 57, 59, and 61, or one that has at least 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%, 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% sequence identity to the amino acid sequence of any one of SEQ ID NOs:47, 49, 51, 53, 55, 57, 59, and 61, or a variant thereof. In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or a derivative thereof is isolated from the host cell, for example, isolated from the extracellular environment of the host cell. In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is spontaneously secreted, completely or partially, from the host cell. In alternative embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is transported into the extracellular environment. In further embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is passively transported or spontaneously secreted into the extracellular environment.
The first polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15, with one or more amino acid substitutions, additions, insertions, or deletions, the second polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 24, 26, 28, 30, 32, 34, 36, and 38, wherein the first and second polypeptides together have alpha-keto acid decarboxylase activity. In certain embodiments, the first polypeptide comprises one or more or all of the amino acid sequence motifs selected from SEQ ID NOs:17-23. The second polypeptide comprises one or more or all of the amino acid sequence motifs selected from SEQ ID NOs:40-46. In some embodiments, the third polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 47, 49, 51, 53, 55, 57, 59, and 61, with one or more amino acid substitutions, additions, insertions, or deletions, wherein the third polypeptide has lipoamide acyltransferase activity. The third polypeptide comprises one or more or all of the amino acid sequence motifs selected from SEQ ID NOs:63-68. In some embodiments, the first, second and third polypeptides are capable of catalyzing the conversion of alpha-keto acids to branched acyl-CoAs. It is within the capacity of those skilled in the art to devise a suitable enzymatic assay using the appropriate substrates. Examples of such assays are described herein.
In some embodiments, the first, second, and third polypeptides independently comprises 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the first and second polypeptides independently comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the third polypeptide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the first and second polypeptides have branched-chain alpha-keto acid decarboxylase activity and the third polypeptide has lipoamide acyltransferase activity. In some embodiments, the first, second and third polypeptides are capable of catalyzing the conversion of branched alpha-keto acids to branched acyl-CoAs.
In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a fourth gene encoding a fourth polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, or one that has 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%, 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% sequence identity to the amino acid sequence of any one of SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, or a variant thereof. In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or a derivative thereof is isolated from the host cell, for example, from the extracellular environment. In certain embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is spontaneously secreted, partially or completely, into the extracellular environment. In other embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is transported into the extracellular environment. In certain embodiments, the branched fatty aldehyde, branched fatty alcohol or the derivative thereof is passively transported into the extracellular environment.
The fourth polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:69, 71, 73, 75, 77, 79, 81, and 83, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has lipoamide dehydrogenase activity. In certain embodiments, the fourth polypeptide comprises one or more or all of amino acid sequence motifs selected from SEQ ID NOs:85-89. In some embodiments, the fourth polypeptide comprises 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the fourth polypeptide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the fourth polypeptide has lipoamide dehydrogenase activity. In some embodiments, the first, second, third and fourth polypeptides have branched chain alpha-keto acid decarboxylase and/or lipoamide acyltransferase and/or lipoamide dehydrogenase activity. In some embodiments, the first, second, third and fourth polypeptides, optionally forming a complex, are capable of catalyzing the conversion alpha-keto acids to branched-chain acyl-CoAs.
In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell further expressing a gene encoding a beta-ketoacyl ACP synthase comprising the amino acid sequence of any one of SEQ ID NOs: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, or one that has 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%, 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% sequence identity to the amino acid sequence of any one of SEQ ID NOs:90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, or a variant thereof. In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or a derivative thereof is isolated from the host cell, for example, from the extracellular environment. In certain embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is spontaneously secreted, partially or completely, into the extracellular environment. In other embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is transported into the extracellular environment. In certain embodiments, the branched fatty aldehyde, branched fatty alcohol, or the derivative thereof is passively transported into the extracellular environment.
The beta ketoacyl-ACP synthase polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, with one or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the beta ketoacyl-ACP synthase polypeptide comprises one or more or all of amino acid sequence motifs selected from SEQ ID NOs:122-127. In some embodiments, the polypeptide has FabH activity. In certain embodiments, the beta ketoacyl-ACP synthase polypeptide has specificity for branched-chain fatty acyl-CoA substrates. In certain embodiments, the polypeptide is capable of catalyzing the condensation reaction between a branched acyl-CoA and malonyl-ACP. It is within the capacity of those skilled in the art to devise a suitable enzymatic assay using the appropriate substrates in order to distinguish those polypeptides having sequence homology to the beta-ketoacyl-ACP synthase polypeptides herein but are not suitable or does not have specificity for branched-chain substrates. Two examples of such enzymatic assays are described herein.
The beta ketoacyl-ACP synthase polypeptide can comprise 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments, the polypeptide comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200 or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide has FabH activity. In some embodiments, the polypeptide has specificity for branched-chain acyl-CoAs. In some embodiments, the polypeptide is capable of catalyzing the condensation of a branched acyl-CoA and malonyl-ACP.
In certain embodiments, the first polypeptide comprises an amino acid sequence motif of any one of or one or more or all of SEQ ID NOs:17-23, wherein the first polypeptide is of about 200 to about 800 amino acid residues in length, or about 300 to about 700 amino acid residues in length, or about 400 to about 600 amino acids in length. In some embodiments, the second polypeptide comprises an amino acid sequence motif of any one of or one or more or all of SEQ ID NOs:40-46, wherein the second polypeptide is about 200 to about 800 amino acid residues in length, or about 300 to about 700 amino acid residues in length, or about 400 to about 600 amino acid residues in length. In some embodiments, the third polypeptide comprises an amino acid sequence motif of any one of or one or more or all of SEQ ID NOs:63-68, wherein the first polypeptide is of about 200 to about 800 amino acid residues in length, or about 300 to about 700 amino acid residues in length, or about 400 to about 600 amino acid residues in length. In some embodiments, the first, second and optionally the third polypeptides are capable of catalyzing the conversion of alpha-keto acid substrates to branched acyl-CoAs.
In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell further expressing a gene encoding a fatty aldehyde biosynthesis polypeptide selected from those listed in the Table 6, or a variant thereof. In some embodiments, the fatty aldehyde biosynthesis polypeptide comprises the amino acid sequence of an enzyme listed in Table 6, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has carboxylic acid reductase activity. In some embodiments, the polypeptide has fatty acid reductase activity. In some embodiments, the fatty aldehyde biosynthesis polypeptide comprises one or more of the following conservative amino acid substitutions. In some embodiments, the fatty aldehyde biosynthesis polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide has carboxylic acid reductase activity. In some embodiments, the polypeptide has fatty acid reductase activity.
In some embodiments, the branched fatty alcohol or a derivative thereof is isolated from the host cell, for example, from the extracellular environment. In some embodiments, the branched fatty alcohol or the derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty alcohol or the derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or the derivative thereof is passively transported into the extracellular environment.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell wherein a gene encoding a fatty acid synthase is modified. For example, modifying the expression of a gene encoding a fatty acid synthase includes expressing a gene encoding a fatty acid synthase in the host cell and/or increasing the expression or activity of an endogenous fatty acid synthase in the host cell. Alternatively, modifying the expression of a gene encoding a fatty acid synthase includes attenuating a gene encoding a fatty acid synthase in the host cell and/or decreasing the expression or activity of an endogenous fatty acid synthase in the host cell. In some embodiments, the fatty acid synthase is a thioesterase. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding a fatty alcohol biosynthesis polypeptide. The fatty alcohol biosynthesis polypeptide is, for example, an alcohol dehydrogenase. In particular embodiments, the fatty alcohol biosynthesis polypeptide is one selected from the enzymes listed in Table 8, or a variant thereof.
In certain other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding another aldehyde biosynthetic polypeptide or an acyl-ACP reductase polypeptide comprising the amino acid sequence of any of the enzymes listed in Table 7, or a variant thereof. In some embodiments, the branched fatty alcohol or derivative thereof is isolated from the host cell, for example, from the extracellular environment. In certain embodiments, the branched fatty alcohol or derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty alcohol or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or derivative thereof is passively transported into the extracellular environment.
The acyl-ACP reductase polypeptide, for example, comprises the amino acid sequence of an enzyme selected from those listed in Table 7, with one or more amino acid substitutions, additions, insertions, or deletions, and the polypeptide has reductase activity. In certain embodiments, the polypeptide is capable of catalyzing the conversion of a suitable biological substrate into an aldehyde. The acyl-ACP reductase polypeptide, for example, comprises one or more conservative amino acid substitutions, or has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or more amino acid substitutions, additions, insertions, or deletions. In some embodiments, the polypeptide has reductase activity. In some embodiments, the polypeptide is capable of catalyzing the conversion of a suitable biological substrate into an aldehyde.
In any of the above-described embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to a wild type host cell. For example, the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC—4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP—01644857. In certain embodiments, the genetically engineered host cell comprises a knockout of one or more genes encoding a fatty acid degradation enzyme, such as the aforementioned acyl-CoA synthase genes. In certain embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is tonA (also known as fhuA). Yet in other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
In yet other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a dehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by a gene listed in Table 1 or Table 2. In some embodiments, the host cell comprises a knockout of fabA or a gene listed in Table 1 or Table 2. In other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of an endogenous ketoacyl-ACP synthase, such as an enzyme encoded by fabB or by a gene listed in Table 3 or Table 4. In certain embodiments, the host cell comprises a knockout of fabB or a gene listed in Table 3 or Table 4. In yet other embodiments, the host cell is genetically engineered to express a modified level of a gene encoding a desaturase enzyme, such as desA.
In certain embodiments, the branched-chain alpha-keto acid dehydrogenase complex polypeptides, the beta ketoacyl-ACP synthase polypeptide, the aldehyde biosynthesis polypeptide, the fatty acid synthase, the acyl-ACP reductase, the alcohol biosynthesis polypeptide, and the fatty acid degradation enzyme polypeptide are each independently obtained from a bacterium, a plant, an insect, a yeast, a fungus, or a mammal. For example, each of the above-mentioned polypeptides is from a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, bacterial cell, or any other organism described herein. In certain embodiments, the branched-chain alpha-keto acid dehydrogenase complex polypeptides can be from a bacterium that uses branched amino acids as carbon source, including, for example, Pseudomonas putida or a Bacillus subtilis. In certain embodiments, the branched-chain alpha-keto acid dehydrogenase complex polypeptide can be from a bacterium that comprises branched fatty acids in its phospholipids, including, for example, a Legionella, Stenotrophomonas, Alteromonas, Flavobacterium, Myxococcus, Bacteroides, Micrococcus, Staphylococcus, Bacillus, Clostridium, Listeria, Lactococcus, or Streptomyces bacterium. In some embodiments, the bacterium is a Leginella pneumophila, Stenotrophomonas maltophilia, Alteromonas macleodii, Flabobacterium phsychrophilum, Myxococcus Xanthus, Bacteroides thetaiotaomicron, Macrococcus luteus, Staphylococcus aureus, Clostridium thermocellum, Listeria monocytogenes, Streptomyces lividans, Streptomyces coelicolor, Streptomyces glaucescens, Streptococcus pneumoniae, Streptomyces peucetius, Streptococcus pyogenes, Escherichia coli, Escherichia coli K-12, Lactococcus lactis ssp. Lactis, Mycobacterium tuberculosis, Enterococcus tuberculosis, Bacillus subtilis, Lactobacillus plantarum. In certain embodiments, suitable fatty aldehyde biosynthesis polypeptides, fatty alcohol biosynthesis polypeptides, acyl-ACP reductases, and other polypeptides of the invention can be from a mycobacterium selected from the group consisting of Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans. In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinispora arenicola, or Clavibacter michiganenesis. In certain further embodiments, the polypeptide of the invention is derived from a cyanobacterium, including, for example, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcus marinus subsp. pastoris str. CCMP 1986 PMM0533, Gloeobacter violaceus PCC7421, Nostoc punctiforme PCC73102, Anabaena variabilis ATCC29413, Synechococcus elongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleus chthonoplastes PCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106, Nodularia spumigena CCY9414, Trichodesmium erythraeum IMS101, Microcystis aeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaera watsonii, Thermosynechococcus elongatus, Gloeobacer violaceus, Cyanobium, or Prochlorococcus marinus. 
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell cultured in the presence of at least one biological substrate for the branched-chain alpha-keto acid dehydrogenase polypeptides, the beta ketoacyl-ACP synthase polypeptide, the aldehyde biosynthesis polypeptide, the acyl-ACP reductase, and/or the alcohol biosynthesis polypeptide. Suitable substrate for the branched-chain alpha-keto acid dehydrogenase polypeptides can include, without limitation, 2-oxo-isovalerate, 2-oxo-isobutyrate, or 2-oxo-3-methyl-valerate.
In another aspect, the invention features a surfactant or detergent composition comprising a microbially produced branched fatty alcohol or a derivative thereof. In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a first polynucleotide that hybridizes to a complement of a nucleotide sequence of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16, or to a fragment thereof, and a second polynucleotide that hybridizes to a complement of a second polynucleotide sequence of any one of SEQ ID NOs:25, 27, 29, 31, 33, 35, 37, and 39. In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a third polynucleotide that hybridizes to a complement of a third nucleotide sequence of any one of SEQ ID NOs:48, 50, 52, 54, 56, 58, 60, and 62, or to a fragment thereof, wherein the first and second polynucleotides encode the first and second polypeptides having branched-chain alpha-keto acid decarboxylase activity, and wherein the third polynucleotide encodes a polypeptide having lipoamide acyltransferase activity. In some embodiments, the first and the second polypeptides, optionally forming a single subunit, optionally together with the third polypeptide, are capable of catalyzing the conversion of branched-chain alpha-keto acids to branched acyl-CoAs.
The first polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16, or to a fragment thereof. The second polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, and 39, or to a fragment thereof. The third polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of any one of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a fourth polynucleotide that hybridizes to a complement of a nucleotide sequence of any one of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84, or to a fragment thereof, wherein the fourth polynucleotide encodes a polypeptide having lipoamide dehydrogenase activity. In some embodiments, the first, second, and optionally the third and/or fourth polypeptides are capable of catalyzing the conversion of branched-chain alpha-keto acids into branched acetyl-CoAs.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a polynucleotide that hybridizes to a complement of a nucleotide sequence of any one of SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 121, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having beta ketoacyl-ACP synthase activity. In some embodiments, the polypeptide is capable of catalyzing the condensation of a branched acyl-CoA with malonyl-ACP. In some embodiments, the polypeptide has FabH activity. In some embodiments, the polypeptide has specificity for branched acyl-CoA substrates. The polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence of any one of SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 121, or to a fragment thereof.
In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is isolated from the host cell, for example, from the extracellular environment. In certain embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is passively transported into the extracellular environment.
In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a polynucleotide that hybridizes to a complement of the nucleotide sequence encoding a fatty aldehyde biosynthesis polypeptide listed in Table 6, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having carboxylic acid reductase activity. In some embodiments, the polypeptide has fatty acid reductase activity.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, wherein the gene encoding a fatty acid synthase is modified. In certain embodiments, modifying the expression of a gene encoding a fatty acid synthase includes expressing a gene encoding a fatty acid synthase in the host cell and/or increasing the expression or activity of an endogenous fatty acid synthase in the host cell. In alternate embodiments, modifying the expression of a gene encoding a fatty acid synthase includes attenuating a gene encoding a fatty acid synthase in the host cell and/or decreasing the expression or activity of an endogenous fatty acid synthase in the host cell. In some embodiments, the fatty acid synthase is a thioesterase. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding a fatty alcohol biosynthesis polypeptide. For example, the fatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. In particular embodiments, the fatty alcohol biosynthesis polypeptide is one selected from those listed in Table 8, or a variant thereof.
In any of the above-described embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to a wild type host cell. In some embodiments, the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC—4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP—01644857. In certain embodiments, the genetically engineered host cell comprises a knockout of one or more genes encoding a fatty acid degradation enzyme, such as the aforementioned acyl-CoA synthase genes. In certain embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is tonA (also known as fhuA). Yet in other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
In yet other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a dehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by a gene listed in Table 1 or Table 2. In some embodiments, the host cell comprises a knockout of fabA or a gene listed in Table 1 or Table 2. In other embodiments, the host cell is genetically engineered to express an attenuated level of a ketoacyl-ACP synthase, such as an enzyme encoded by fabB or by a gene listed in Table 3 or Table 4. In certain embodiments, the host cell comprises a knockout of fabB or a gene listed in Table 3 or Table 4. In yet other embodiments, the host cell is genetically engineered to express a modified level of a gene encoding a desaturase enzyme, such as desA.
In some embodiments, the branched fatty alcohol or a derivative thereof is isolated from the host cell, for example, from the extracellular environment. In certain embodiments, the branched fatty alcohol or the derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty alcohol or the derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or the derivative thereof is passively transported into the extracellular environment.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a polynucleotide that hybridizes to a complement of a nucleotide sequence encoding an acyl-ACP reductases selected from those listed in Table 7, or to a fragment thereof, wherein the polynucleotide encodes a polypeptide having reductase activity. The polynucleotide hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions, to a complement of the nucleotide sequence encoding an acyl-ACP reductases selected from those listed in Table 7, or to a fragment thereof.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding a fatty alcohol biosynthesis polypeptide in the host cell. For example, the fatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. In particular embodiments, the fatty alcohol biosynthesis polypeptide is one selected from those listed in Table 8, or a variant thereof.
In any of the above-described embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to a wild type host cell. In some embodiments, the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC—4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP—01644857. In certain embodiments, the genetically engineered host cell comprises a knockout of one or more genes encoding a fatty acid degradation enzyme, such as the aforementioned acyl-CoA synthase genes. In certain embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is tonA (also known as fhuA). Yet in other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
In yet other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a dehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by a gene listed in Table 1 or Table 2. In some embodiments, the host cell comprises a knockout of fabA or a gene listed in Table 1 or Table 2. In other embodiments, the host cell is genetically engineered to express an attenuated level of a ketoacyl-ACP synthase, such as an enzyme encoded by fabB or by a gene listed in Table 3 or Table 4. In certain embodiments, the host cell comprises a knockout of fabB or a gene listed in Table 3 or Table 4. In yet other embodiments, the host cell is genetically engineered to express a modified level of a gene encoding a desaturase enzyme, such as desA.
In some embodiments, the branched fatty alcohol or derivative thereof is isolated from the host cell, e.g., from the extracellular environment. In certain embodiments, the branched fatty alcohol or derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty alcohol or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or derivative thereof is passively transported into the extracellular environment.
In some embodiments, the branched-chain alpha-keto acid dehydrogenase complex, the beta ketoacyl-ACP synthase polypeptide, the aldehyde biosynthesis polypeptide, the fatty acid synthase, the acyl-ACP reductase, the alcohol biosynthesis polypeptide, and the fatty acid degradation enzyme polypeptide are each independently from a bacterium, a plant, an insect, a yeast, a fungus, or a mammal. For example, the branched-chain alpha-keto acid dehydrogenase complex polypeptides can be from a bacterium that uses branched amino acids as carbon source, including, for example, Pseudomonas putida, or Bacillus subtilis. In another example, the branched-chain alpha-keto acid dehydrogenase complex polypeptide can be from a bacterium that comprises branched fatty acids in its phospholipids, including, for example, a Legionella, Stenotrophomonas, Alteromonas, Flavobacterium, Myxococcus, Bccteroides, Micrococcus, Staphylococcus, Bacillus, Clostridium, Listeria, Lactococcus, or Streptomyces bacterium. In some embodiments, the bacterium is a Leginella pneumophila, Stenotrophomonas maltophilia, Alteromonas macleodii, Flabobacterium phsychrophilum, Myxococcus Xanthus, Bacteroides thetaiotaomicron, Macrococcus luteus, Staphylococcus aureus, Clostridium thermocellum, Listeria monocytogenes, Streptomyces lividans, Streptomyces coelicolor, Streptomyces glaucescens, Streptococcus pneumoniae, Streptomyces peucetius, Streptococcus pyogenes, Escherichia coli, Escherichia coli K-12, Lactococcus lactis ssp. Lactis, Mycobacterium tuberculosis, Enterococcus tuberculosis, Bacillus subtilis, Lactobacillus plantarum. In some embodiments, suitable fatty aldehyde biosynthesis polypeptides, fatty alcohol biosynthesis polypeptides, acyl-ACP reductases, and other polypeptides of the invention can be from a mycobacterium selected from the group consisting of Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium ulcerans. In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinispora arenicola, or Clavibacter michiganenesis. In yet further embodiments, the polypeptide of the invention is derived from a cyanobacterium, including, for example, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcus marinus subsp. pastoris str. CCMP1986 PMM0533, Gloeobacter violaceus PCC7421, Nostoc punctiforme PCC73102, Anabaena variabilis ATCC29413, Synechococcus elongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleus chthonoplastes PCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106, Nodularia spumigena CCY9414, Trichodesmium erythraeum IMS101, Microcystis aeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaera watsonii, Thermosynechococcus elongatus, Gloeobacer violaceus, Cyanobium, or Prochlorococcus marinus. 
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell cultured in the presence of at least one biological substrate for the branched-chain alpha-keto acid dehydrogenase polypeptides, the beta ketoacyl-ACP synthase polypeptide, the aldehyde biosynthesis polypeptide, the acyl-ACP reductase, or the alcohol biosynthesis polypeptide. In some embodiments, the host cell is cultured under conditions that allow the expression of the branched-chain alpha-keto acid dehydrogenase polypeptides, the beta ketoacyl-ACP synthase, the aldehyde biosynthesis polypeptide, the acyl-ACP reductase, and/or the alcohol biosynthesis polypeptide. In particular embodiments, the host cell is cultured under conditions that allow the production of branched fatty alcohols or derivatives thereof.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell cultured in the presence of at least one biological substrate for the branched-chain alpha-keto acid dehydrogenase complex, the aldehyde biosynthesis polypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACP reductase polypeptide. Accordingly, the host cell is cultured under conditions that allow expression of branched-chain alpha-keto acid dehydrogenase complex, the aldehyde biosynthesis polypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACP reductase polypeptide.
In some embodiments, the branched fatty alcohol or derivative thereof is isolated from the host cell, e.g., from the extracellular environment. In some embodiments, the branched fatty alcohol or derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty alcohol or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or derivative thereof is passively transported into the extracellular environment.
In another aspect, the invention features a surfactant or detergent composition comprising a microbially produced branched fatty alcohol or a derivative thereof. In certain embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing one or more recombinant vectors comprising at least the E1 alpha and beta subunits of a branched-chain alpha-keto acid dehydrogenase. In certain embodiments, the recombinant vector further comprises an E2 subunit of a branched-chain alpha-keto acid dehydrogenase. The subunits can be introduced into the host cell in separate vectors or together in a single vector. For example, the vector can comprise a first polynucleotide sequence having at least about 30%, e.g., 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 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%, at least about 99%, or 100% identity sequence identity to a polynucleotide sequence listed in FIG. 2A, and a second polynucleotide sequence having at least about 30%, e.g., 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 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%, at least about 99%, or 100% identity sequence identity to a polynucleotide sequence listed in FIG. 2B. In another example, the vector can further comprise a third polynucleotide having at least about 30%, e.g., 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 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%, at least about 99%, or 100% identity sequence identity to a polynucleotide sequence listed in FIG. 2C. The polynucleotides encoding the alpha and beta subunits of the E1 subunit can be linked and constitute a single operon, or they may be separately introduced into a vector and/or into a host cell. Likewise, the polynucleotides encoding the E1 subunit and the polynucleotide encoding the E2 subunit can be linked and constitute a single operon, or they may be separately introduced into a vector and/or into a host cell. For example, a first vector can comprise a first polynucleotide sequence having at least about 30%, e.g., 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 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%, at least about 99%, or 100% identity sequence identity to a polynucleotide sequence listed in FIG. 2A, and a second vector can comprise a second polynucleotide having at least about 30%, e.g., 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 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%, at least about 99%, or 100% identity sequence identity to a polynucleotide sequence listed in FIG. 2B.
In some embodiment, the nucleotide sequence of the first polynucleotide has 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%, 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% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16; the second polynucleotide has 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%, 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% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, and 39; and the nucleotide sequence of the third polynucleotide has 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%, 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% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62. In some embodiment, the nucleotide sequence of the first polynucleotide is any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16, the nucleotide sequence of the second polynucleotide is any one of SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, and 39, and the nucleotide sequence of the third polynucleotide, when present, is any one of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62.
In some embodiment, each of the vectors above, or another vector can comprise a fourth polynucleotide sequence having at least about 30%, e.g., 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 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%, at least about 99%, or 100% sequence identity to a polynucleotide sequence listed in FIG. 2D. In certain embodiments, the nucleotide sequence of the fourth polynucleotide has 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%, 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% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84. In some embodiments, the nucleotide sequence of the fourth polynucleotide is any one of SEQ ID NOs:70, 72, 74, 76, 78, 80, 82, and 84.
In some embodiments, each of the vectors above, or another vector can be introduced into the host cell wherein the vector comprises a beta-ketoacyl ACP synthase nucleotide that has at least about 30%, e.g., 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 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%, at least about 99%, or 100% sequence identity to a polynucleotide sequence listed in FIG. 2E. In certain embodiments, the nucleotide sequence of the beta-ketoacyl ACP synthase nucleotide has 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%, 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% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 121. In some embodiments, the nucleotide sequence of beta-ketoacyl ACP synthase nucleotide is SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 121.
In yet another embodiment, an individual vector comprising a beta-ketoacyl-ACP synthase nucleotide that has at least about 30%, e.g., 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 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%, at least about 99%, or 100% sequence identity to a polynucleotide sequence listed in FIG. 2E can be introduced into a suitable host cell, independent of whether one or more other vectors comprising one or more subunits of a branched-chain alpha-keto acid dehydrogenase is introduced into the same cell. For example, the host cell can suitably be one that expresses an endogenous branched-chain alpha-keto acid dehydrogenase, or one or more subunits thereof.
In some embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is isolated from the host cell, for example, from the extracellular environment. In some embodiments, the branched fatty aldehyde, branched fatty alcohol or derivative thereof is spontaneously secreted, partially or completely, from the host cell. In alternative embodiments, the branched fatty aldehyde, branched fatty alcohol or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty aldehyde, branched fatty alcohol, or derivative thereof is passively transported into the extracellular environment.
The recombinant vector can further comprises a promoter operably linked to the nucleotide sequence. In certain embodiments, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
In other embodiments, the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence.
In some embodiments, the recombinant vector is a plasmid.
In some embodiments, the host cell expresses a polypeptide encoded by the recombinant vector. In some embodiments, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region. In an exemplary embodiment, one or more of the polynucleotides encoding a branched-chain alpha-keto acid dehydrogenase polypeptide, a beta ketoacyl-ACP synthase polypeptide, a fatty aldehyde biosynthesis polypeptide, a fatty alcohol biosynthesis polypeptide, and/or an acyl-ACP reductase of the invention can be stably incorporated into the genomic DNA of the host cell, and the expression of the polynucleotide sequence is under the control of a regulated promoter region.
In some embodiment, an above-described vector or another vector can be introduced into the host cell wherein the vector comprises a fatty aldehyde biosynthesis polynucleotide having at least about 70% sequence identity to a nucleotide sequence encoding an enzyme listed in Table 6.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell wherein the expression of a gene encoding a fatty acid synthase is modified. In certain embodiments, modifying the expression of a gene encoding a fatty acid synthase includes expressing a gene encoding a fatty acid synthase in the host cell and/or increasing the expression or activity of an endogenous fatty acid synthase in the host cell. In alternate embodiments, modifying the expression of a gene encoding a fatty acid synthase includes attenuating a gene encoding a fatty acid synthase in the host cell and/or decreasing the expression or activity of an endogenous fatty acid synthase in the host cell. In some embodiments, the fatty acid synthase is a thioesterase. In particular embodiments, the thioesterase is encoded by tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell expressing a gene encoding a fatty alcohol biosynthesis polypeptide. For example, the fatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. In particular embodiments, the fatty alcohol biosynthesis polypeptide comprises the amino acid sequence of an enzyme listed in Table 8, or a variant thereof.
In any of the embodiments described above, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a fatty acid degradation enzyme relative to a wild type host cell. In some embodiments, the host cell is genetically engineered to express an attenuated level of an acyl-CoA synthase relative to a wild type host cell. In particular embodiments, the host cell expresses an attenuated level of an acyl-CoA synthase encoded by fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC—4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP—01644857. In certain embodiments, the genetically engineered host cell comprises a knockout of one or more genes encoding a fatty acid degradation enzyme, such as the aforementioned acyl-CoA synthase genes. In certain embodiments, the host cell is genetically engineered to express, relative to a wild type host cell, a decreased level of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In some embodiments, the gene encoding an acyl-CoA dehydrogenase is fadE. In some embodiments, the gene encoding an outer membrane protein receptor is tonA (also known as fhuA). Yet in other embodiments, the gene encoding a transcriptional regulator of fatty acid biosynthesis is fabR.
In yet other embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell, which is genetically engineered to express an attenuated level of a dehydratase/isomerase enzyme, such as an enzyme encoded by fabA or by a gene listed in Table 1 or Table 2. In some embodiments, the host cell comprises a knockout of fabA or a gene listed in Table 1 or Table 2. In other embodiments, the host cell is genetically engineered to express an attenuated level of a ketoacyl-ACP synthase, such as an enzyme encoded by fabB or by a gene listed in Table 3 or Table 4. In certain embodiments, the host cell comprises a knockout of fabB or a gene listed in Table 3 or Table 4. In yet other embodiments, the host cell is genetically engineered to express a modified level of a gene encoding a desaturase enzyme, such as desA.
In certain other embodiments, any of the vectors comprising the E1 alpha, E1 beta, and/or optionally E2 and/or optionally E3 subunits of a branched-chain alpha-keto acid dehydrogenase complex or another vector can be introduced into the host cell wherein the vector further comprises an acyl-ACP reductase polynucleotide having at least about 70% sequence identity to a nucleotide sequence encoding an enzyme listed in Table 7.
In some embodiments, the host cell is cultured in the presence of at least one biological substrate for the branched-chain alpha-keto acid dehydrogenase complex, the aldehyde biosynthesis polypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACP reductase polypeptide. In certain embodiments, the host cell is cultured under conditions that are sufficient for expressing a branched-chain alpha-keto acid dehydrogenase complex, an aldehyde biosynthesis polypeptide, an alcohol biosynthesis polypeptide, and/or an acyl-ACP reductase polypeptide. In certain other embodiments, the host cell is cultured under conditions that allow the production of branched fatty alcohols or derivatives thereof.
In some embodiments, the microbially produced branched fatty alcohol and/or derivative thereof is produced by a host cell cultured in the presence of at least one biological substrate for the branched-chain alpha-keto acid dehydrogenase complex, the aldehyde biosynthesis polypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACP reductase polypeptide. Accordingly, the host cell is cultured under conditions that allow expression of branched-chain alpha-keto acid dehydrogenase complex, the aldehyde biosynthesis polypeptide, the alcohol biosynthesis polypeptide, and/or the acyl-ACP reductase polypeptide.
In some embodiments, the branched fatty alcohol or derivative thereof is isolated from the host cell, for example, from the extracellular environment. In some embodiments, the branched fatty alcohol or derivative thereof is secreted from the host cell. In alternative embodiments, the branched fatty alcohol or derivative thereof is transported into the extracellular environment. In other embodiments, the branched fatty alcohol or derivative thereof is passively transported into the extracellular environment.
In any of the aspects of the invention described herein, the host cell can be selected from the group consisting of a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, and bacterial cell. In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell. In some embodiments, the host cell is selected from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. 
In certain embodiments, the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
In other embodiments, the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.
In yet other embodiments, the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.
In yet other embodiments, the host cell is an Actinomycetes cell.
In some embodiments, the host cell is a Saccharomyces cerevisiae cell.
In particular embodiments, the host cell is a cell from an eukaryotic plant, algae, cyanolacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, engineered organisms thereof, or a synthetic organism. In some embodiments, the host cell is light dependent or fixes carbon. In some embodiments, the host cell is light dependent or fixes carbon. In some embodiments, the host cell has autotrophic activity. In some embodiments, the host cell has photoautotrophic activity, such as in the presence of light. In some embodiments, the host cell is heterotrophic or mixotrophic in the absence of light. In certain embodiments, the host cell is a cell from Avabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chloroflexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis. 
In other embodiments, the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell.
In yet other embodiments, the host cell is an E. coli cell. In certain embodiments, the E. coli cell is a strain B, a strain C, a strain K, or a strain W E. coli cell.
In further embodiments, the host cell can be genetically engineered to express an attenuated level of a dehydratase/isomerase enzyme. For example, an E. coli cell is chosen as a suitable host cell, wherein one or more of the endogenous dehydratase/isomerase enzymes such as those listed in Table 1 below can be attenuated or knocked out.







TABLE 1







E. coli dehydratase/isomerase enzymes












Polynucleotide
Polypeptide


Gene
Name
Acc. No.
Acc. No.





fabA
beta-hydroxydecanoyl
GU072596.1
ACY27485.1



thioester dehydrase


fabZ
(3R)-hydroxymyristol acyl
GU072604
ACY27493.1



carrier protein dehydratase


cysM
cysteine synthase B
CP001637.1
ACX38914


maoC
fused aldehyde dehydrogenase/
CP001637
ACX39905.1



enoyl-CoA hydratase









Other dehydratase/isomerase enzymes encoded by a gene listed below in Table 2 can also be attenuated or knocked from an organism comprising such a gene.







TABLE 2







Other dehydatase/isomerase enzymes








Organism
Accession No.





Shigella sp. D9
ZP_05432652


Citrobacter youngae ATCC 29220
ZP_04561391.1


Salmonella enterica
YP_001570967.1


Escherichia fergusonii ATCC 35469
YP_002382254.1


Klebsiella pneumoniae NTUH-K2044
YP_002918743.1


Enterobacter cancerogenus ATCC 35316
ZP_03281954.1


Cronobacter turicensis
CBA29728.1


Erwinia pyrifoliae Ep1/96
YP_002649242.1


Pectobacterium carotovorum subsp. carotovorum PC1
YP_003018119.1


Dickeya dadantii Ech703
YP_002987184.1


Edwardsiella ictaluri 93-146
YP_002932813.1


Providencia alcalifaciens DSM 30120
ZP_03317956.1


Yersinia kristensenii ATCC 33638
ZP_04624337.1


Photorhabdus asymbiotica
YP_003041580.1


Pantoea sp. At-9b
ZP_05728924.1


Actinobacillus succinogenes 130Z
YP_001344737.1


Mannheimia succiniciproducens MBEL55E
YP_088386.1


Pasteurella multocida subsp. multocida str. Pm70
NP_245421.1


Haemophilus somnus 129PT
YP_719117.1


Proteus mirabilis HI4320
YP_002150544.1


Sodalis glossinidius str. ‘morsitans’
YP_454706.1


Candidatus Blochmannia pennsylvanicus str. BPEN
YP_277927.1


Aggregatibacter aphrophilus NJ8700
YP_003007342.1


Vibrio cholerae MZO-3
ZP_01958381.1


Baumannia cicadellinicola str. Hc
YP_588853.1


(Homalodisca coagulata)


Vibrionales bacterium SWAT-3
ZP_01815187.1


Aliivibrio salmonicida LFI1238
YP_002262988.1


Aeromonas salmonicida subsp. salmonicida A449
YP_001141819.1


Wigglesworthia glossinidia endosymbiont
NP_871303.1


of Glossina brevipalpis


Glaciecola sp. HTCC2999
ZP_03560821.1


Alteromonas macleodii ATCC 27126
ZP_04714556.1









In other embodiments, the host cell is genetically engineered to express an attenuated level of an endogenous ketoacyl-ACP synthase. For example, an E. coli cell is used as a suitable host cell, wherein one or more of the ketoacyl-ACP genes listed in Table 3 below can be attenuated or knocked out.







TABLE 3







E. coli ketoacyl-ACP synthase enzymes












Polynucleotide
Polypeptide


Gene
Name
Acc. No.
Acc. No.





fabB
B-ketoacyl synthase/
GU072597.1
ACY27486.1



3-oxoacyl-[acyl-carrier-protein]



synthase I


fabF
3-oxoacyl-[acyl-carrier-
GU072598.1
ACY27487



protein] synthase II


fadJ
fused enoyl-CoA hydratase
CP001637.1
ACX38989.1



and epimerase/isomerase/



3-hydroxyacyl-CoA



dehydrogenase


xerC
site-specific tyrosine
CP001637.1
ACX41768.1



recombinase


yqeF
predicted acyltransferase
CP001637.1
ACX38529.1


murQ
predicted PTS component
CP001637.1
ACX38907.1









Other endogenous ketoacyl-ACP synthases, such as the ones listed in Table 4, can be attenuated or knocked out from an organism comprising such an enzyme.







TABLE 4







Other ketoacyl-ACP synthases








Organism
Accession No.





Shigella boydii CDC 3083-94
YP_001881145.1


Escherichia fergusonii ATCC 35469
YP_002382013.1


Salmonella enterica subsp. arizonae
YP_001569590.1


Citrobacter sp. 30_2
ZP_04562837.1


Klebsiella pneumoniae subsp. pneumoniae MGH
YP_001336360.1


78578


Pectobacterium carotovorum subsp. carotovorum
ZP_03831287.1


WPP14


Enterobacter cancerogenus ATCC 35316
ZP_03283474.1


Pantoea sp. At-9b
ZP_05730617.1


Cronobacter turicensis
CBA32510.1


Dickeya dadantii Ech586
ZP_05723897.1


Erwinia tasmaniensis Et1/99
YP_001907100.1


Serratia proteamaculans 568
YP_001479594.1


Edwardsiella ictaluri 93-146
YP_002934130.1


Sodalis glossinidius str. ‘morsitans’
YP_455303.1


Yersinia aldovae ATCC 35236
ZP_04620215.1


Providencia stuartii ATCC 25827
ZP_02961167.1


Photorhabdus asymbiotica
YP_003040275.1


Proteus mirabilis HI4320
YP_002151524.1


Candidatus Blochmannia pennsylvanicus str. BPEN
YP_278005.1


Glaciecola sp. HTCC2999
ZP_03561088.1


Vibrio cholerae V51
ZP_04919940.1


Wigglesworthia glossinidia endosymbiont
NP_871411.1


of Glossina brevipalpis


Tolumonas auensis DSM 9187
YP_002892770.1


Actinobacillus pleuropneumoniae serovar 1 str. 4074
ZP_00134992.2


Aggregatibacter aphrophilus NJ8700
YP_003007711.1


Pseudoalteromonas tunicata D2
ZP_01135065.1


Vibrionales bacterium SWAT-3
ZP_01816638.1


Pasteurella multocida subsp. multocida str. Pm70
NP_245276.1


Mannheimia succiniciproducens MBEL55E
YP_088783.1


Haemophilus somnus 129PT
YP_718877.1


Shewanella loihica PV-4
YP_001094535.1


Aliivibrio salmonicida LFI1238
YP_002262558.1









In yet other embodiments, the host cell is genetically engineered to express a modified level of a gene encoding a desaturase enzyme, such as desA.
In certain embodiments, the microorganism is genetically engineered to express a modified level (including, e.g., to attenuate or knock out or to express or overexpress) of a gene encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments, the fatty aldehyde biosynthesis polypeptide comprises an amino acid sequence that has at least 70% sequence identity to an enzyme listed in Table 6.
In certain embodiments, the microorganism is genetically engineered to express a modified level of a fatty acid synthase in the host cell. An exemplary fatty acid synthase is a thioesterase encoded by, for example, tesA, tesA without leader sequence, tesB, fatB, fatB2, fatB3, fatA, or fatA1.
In certain embodiments, the microorganism is genetically engineered to express a modified level of gene encoding a fatty alcohol biosynthesis polypeptide. For example, the fatty alcohol biosynthesis polypeptide is an alcohol dehydrogenase. In particular embodiments, the fatty alcohol biosynthesis polypeptide comprises an amino acid sequence that has at least 70% sequence identity to an enzyme listed in Table 8.
Branched-Chain Alpha-Keto Acid Dehydrogenase Complex (BKD Complex) and Beta Ketoacyl-ACP Synthase
The methods described herein can be used to produce branched fatty alcohols and/or derivatives, for example, from alpha keto acids. The oxidative decarboxylation step, which converts the alpha keto acids to the corresponding branched-chain acyl-CoA involves a branched-chain α-keto acid dehydrogenase complex (bkd; EC 1.2.4.4.) (Denoya et al., J. Bacteria 177:3504 (1995)), which consists of E1 alpha/beta (decarboxylase), E2 (dihydrolipoyl transacylase), and E3 (dihydrolipoyl dehydrogenase) subunits. Any microorganism that possesses branched-chain fatty acids, and/or grows on branched-chain amino acids can be used as a source to isolate bkd genes for expression in host cells, for example, E. coli. Furthermore, E. coli has the E3 component as part of its pyruvate dehydrogenase complex (lpd, EC 1.8.1.4, GenBank accession NP—414658). Thus, branched fatty alcohols and/or derivatives can be made by heterologously expressing only the E1 alpha/beta and E2 bkd genes. Furthermore, certain of the host cells, including E. coli, can produce branched products when only the E1 alpha/beta is expressed without co-expression of the E2 bkd gene.
On the other hand, microorganisms that endogenously express a suitable beta-ketoacyl ACP synthase can be engineered to express or overexpress at least the first (E1) subunit of a branched-chain alpha keto acid dehydrogenase complex, optionally also the second (E2) and/or the third (E3) subunits of that complex to produce the desirable branched fatty alcohols and/or derivatives thereof. The endogenous beta-ketoacyl ACP synthase can be overexpressed, or can be modified such that it is attenuated or deleted, and a heterologous beta-ketoacyl ACP synthase gene can be expressed in its place.
In a further embodiment, microorganisms that endogenously express at least the first (E1) subunit of a branched-chain alpha keto acid dehydrogenase complex, and optionally also the second (E2) and/or the third (E3) subunits of that complex, can be engineered to express or overexpress a beta-ketoacyl ACP synthase. For example, the endogenous genes encoding the subunits of the branched-chain alpha keto acid dehydrogenase complex can be overexpressed, or can be modified such that they are attenuated or deleted and a gene encoding one or more subunits of a heterologous branched-chain alpha keto acid dehydrogenase complex can be expressed in the host cell.
Substrates for Branched Fatty Alcohol Production
The branched fatty alcohols and/or derivatives, as well as the surfactant compositions comprising them, can be produced from, for example, branched fatty aldehydes, which themselves can be produced from an appropriate substrate. While not wishing to be bound by theory, it is believed that the branched fatty aldehyde biosynthetic polypeptides described herein produce branched fatty aldehydes from substrates via a reduction mechanism. In some instances, the substrate is a branched fatty acid derivative, and a fatty aldehyde having particular branching patterns and carbon chain length can be produced from a branched fatty acid derivative having those characteristics. The branched fatty aldehyde can then be converted into the desired branched fatty alcohol in a reaction catalyzed by a fatty alcohol biosynthesis polypeptide.
Alternatively, a suitable acyl-ACP reductases can be employed to convert a branched acyl-ACP into a fatty aldehyde, which can in turn be converted into a branched fatty alcohol in a reaction catalyzed by a fatty alcohol biosynthesis polypeptide.
Accordingly, each step within a biosynthetic pathway that leads to the production of a branched fatty acid derivative substrate can be modified to produce or overproduce the branched substrate of interest. For example, known genes involved in the fatty acid biosynthetic pathway or the fatty aldehyde biosynthesis pathway can be expressed, overexpressed, or attenuated in host cells to produce a desired substrate (see, e.g., International Publication WO 2008/119082, the disclosure of which is incorporated by reference).
Synthesis of Branched Fatty Alcohols and Substrates
Fatty acid synthase (FAS) is a group of polypeptides that catalyze the initiation and elongation of acyl chains (Marrakchi et al., Biochemical Society, 30: 1050-1055 (2002)). The acyl carrier protein (ACP) along with the enzymes in the FAS pathway control the length, degree of saturation, and branching of the fatty acid derivatives produced. The fatty acid biosynthetic pathway involves the precursors acetyl-CoA and malonyl-CoA. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (fab) and acetyl-CoA carboxylase (acc) gene families (see, e.g., Heath et al., Prog. Lipid Res., 40(6): 467-97 (2001)).
Host cells can be engineered to express fatty acid derivative substrates by recombinantly expressing or overexpressing one or more fatty acid synthase genes, such as acetyl-CoA and/or malonyl-CoA synthase genes. For example, to increase acetyl-CoA production, one or more of the following genes can be expressed in a host cell: pdh (a multienzyme complex comprising aceEF (which encodes the E1p dehydrogenase component, the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes, and lpd), panK, fabH, fabB, fabD, fabG, acpP, and fabF. Exemplary GenBank accession numbers for these genes are: pdh (BAB34380, AAC73227, AAC73226), panK (also known as CoA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabB (P0A953), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), and fabF (AAC74179). Additionally, the expression levels of fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA, and/or ackB can be attenuated or knocked-out in an engineered host cell by transformation with conditionally replicative or non-replicative plasmids containing null or deletion mutations of the corresponding genes or by substituting promoter or enhancer sequences. Exemplary GenBank accession numbers for these genes are: fadE (AAC73325), gspA (AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430). The resulting host cells will have increased acetyl-CoA production levels when grown in an appropriate environment.
Malonyl-CoA overexpression can be affected by introducing accABCD (e.g., accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acid production can be further increased by introducing into the host cell a DNA sequence encoding a lipase (e.g., accession numbers CAA89087, CAA98876).
In addition, inhibiting PlsB can lead to an increase in the levels of long chain acyl-ACP, which will inhibit early steps in the pathway (e.g., accABCD, fabH, and fabI). The plsB (e.g., accession number AAC77011) D311E mutation can be used to increase the amount of available fatty acids.
In addition, a host cell can be engineered to overexpress a sfa gene (suppressor of fabA, e.g., accession number AAN79592) to increase production of monounsaturated fatty acids (Rock et al., J. Bacteriology, 178: 5382-5387 (1996)).
The chain length of a fatty acid derivative substrate can be selected for by modifying the expression of selected thioesterases. Thioesterase influences the chain length of fatty acids produced. Hence, host cells can be engineered to express, overexpress, have attenuated expression, or not to express one or more selected thioesterases to increase the production of a preferred fatty acid derivative substrate. For example, C10 fatty acids can be produced by expressing a thioesterase that has a preference for producing C10 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C10 fatty acids (e.g., a thioesterase which prefers to produce C14 fatty acids). This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 10. In other instances, C14 fatty acids can be produced by attenuating endogenous thioesterases that produce non-C14 fatty acids and expressing the thioesterases that have a preference for C14-ACP. In some situations, C12 fatty acids can be produced by expressing thioesterases that have a preference for C12-ACP and attenuating thioesterases that preferentially produce non-C12 fatty acids. Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, or GC-MS subsequent to cell lysis. Non-limiting examples of thioesterases that can be used in the methods described herein are listed in Table 5.







TABLE 5







Thioesterases









Accession Number
Source Organism
Gene





AAC73596
E. coli
tesA without




leader sequence


AAC73555
E. coli
tesB


Q41635, AAA34215
Umbellularia california
fatB


AAC49269
Cuphea hookeriana
fatB2


Q39513; AAC72881
Cuphea hookeriana
fatB3


Q39473, AAC49151
Cinnamonum camphorum
fatB


CAA85388
Arabidopsis thaliana
fatB [M141T]*


NP 189147; NP 193041
Arabidopsis thaliana
fatA


CAC39106
Bradyrhiizobium japonicum
fatA


AAC72883
Cuphea hookeriana
fatA


AAL79361
Helianthus annus
fatA1





*Mayer et al., BMC Plant Biology, 7: 1-11 (2007)






In certain embodiments, a host cell, which is used to produce branched fatty alcohols and/or derivatives herein, can be engineered to express or overexpress one of more fatty aldehyde biosynthetic polypeptides. Alternatively, the host cell can be engineered to express an attenuated level of an endogenous fatty aldehyde biosynthetic polypeptide. In other instances, a fatty aldehyde biosynthetic polypeptide, a variant, or a fragment thereof is expressed in a host cell that contains a naturally occurring mutation that results in an increased level of branched fatty aldehyde substrate in the host cell or of branched fatty alcohol produced by the host cell. In some instances, a branched fatty aldehyde is produced by expressing a fatty aldehyde biosynthesis gene, for example, a carboxylic acid reductases gene, encoding a protein listed in Table 6, below, as well as a polynucleotide variant there. In some instances, the fatty aldehyde biosynthesis gene encodes one of the enzymes listed in Table 6 below.







TABLE 6







Fatty Aldehyde Biosynthesis Genes








Name/Organism
Accession No.





Nocardia sp. NRRL 5646
>gi|40796035|gb|AAR91681.1|


Mycobacterium tuberculosis
>gi|15609727|ref|NP_217106.1


H37Rv


Mycobacterium smegmatis
>gi|118174788|gb|ABK75684.1|


str. MC2 155


Mycobacterium smegmatis
>gi|118469671|ref|YP_889972.1|


str. MC2 155


FadD9
uniprot|A0PPD8|A0PPD8_MYCUA


Tsukamurella paurometabola
>gi|227980601|ref|ZP_04027864.1|


DSM 20162


Cyanobium sp. PCC 7001
>gi|254431429|ref|ZP_05045132.1|


Putative acyl-CoA
>uniprot|A0QIB5|A0QIB5_MYCA1


dehydrogenase


NAD dependent epimerase/
>uniprot|A0QWI7|A0QWI7_MYCS2


dehydratase


Mycobacterium intracellulare
>gi|254819907|ref|ZP_05224908.1|


ATCC13950


Putative long-chain fattyacid-
>uniprot|A0R484|A0R484_MYCS2


CoA ligase


Mycobacterium kansasii
>gi|240173202|ref|ZP_04751860.1|


ATCC 12478


Probable fatty-acid-CoA
>uniprot|A1KLT8|A1KLT8_MYCBP


ligase fadD9


Mycobacterium intracellulare
>gi|254822803|ref|ZP_05227804.1|


ATCC13950


Fatty-acid-CoA ligase fadD9
>uniprot|A1QUM2|A1QUM2_MYCTF


Thioester reductase domain
>uniprot|A1T887|A1T887_MYCVP


Thioester reductase domain
>uniprot|A1UFA8|A1UFA8_MYCSK


Mycobacterium avium subsp.
>gi|254775919|ref|ZP_05217435.1|


ATCC 25291


Thioester reductase domain
>uniprot|A3PYW9|A3PYW9_MYCSJ


Mycobacterium leprae Br4923
>gi|219932734|emb|CAR70557.1|


Putative acyl-CoA synthetase
>uniprot|A5CM59|A5CM59_CLAM3


Thioester reductase domain
>uniprot|A8M8D3|A8M8D3_SALAI


Probable fatty-acid-CoA ligase
>uniprot|B1MCR9|B1MCR9_MYCAB


FadD


Probable fatty-acid-CoA ligase
>uniprot|B1MCS0|B1MCS0_MYCAB


FadD


Putative fatty-acid-CoA ligase
>uniprot|B1MDX4|B1MDX4_MYCAB


Probable fatty-acid-coa ligase
>uniprot|B1MLD7|B1MLD7_MYCAB


FadD


Putative carboxylic acid
>uniprot|B1VMZ4|B1VMZ4_STRGG


reductase


Fatty-acid-CoA ligase FadD9_1
>uniprot|B2HE95|B2HE95_MYCMM


Fatty-acid-CoA ligase FadD9
>uniprot|B2HN69|B2HN69_MYCMM


Putative Acyl-CoA synthetase
>uniprot|O69484|O69484_MYCLE


Probable peptide synthetase nrp
>uniprot|Q10896|Q10896_MYCTU


Putative carboxylic acid
>uniprot|Q5YY80|Q5YY80_NOCFA


reductase


ATP/NADPH-dependent
>uniprot|Q6RKB1|Q6RKB1_9NOCA


carboxylic acid reductase


FadD9
>uniprot|Q741P9|Q741P9_MYCPA


Substrate--CoA ligase, putative
>uniprot|Q7D6X4|Q7D6X4_MYCTU


Probable fatty-acid-coa ligase
>uniprot|Q7TY99|Q7TY99_MYCBO


fadd9


Putative acyl-CoA synthetase
>uniprot|Q9CCT4|Q9CCT4_MYCLE


Putative uncharacterized protein
>uniprot|Q54JK0|Q54JK0_DICDI


Putative non-ribosomal peptide
>uniprot|Q2MFQ3|Q2MFQ3_STRRY


synthetase


Mycobacterium tuberculosis
>gi|215431545|ref|ZP_03429464.1|


EAS054


Mycobacterium tuberculosis
>gi|218754327|ref|ZP_03533123.1|


GM 1503


Mycobacterium tuberculosis
>gi|215446840|ref|ZP_03433592.1|


T85


Mycobacterium tuberculosis
>gi|219558593|ref|ZP_03537669.1|


T17


Mycobacterium intracellulare
>gi|254819907|ref|ZP_05224908.1|


ATCC13950









In certain embodiments, a host cell, which is used to produce branched tatty alcohols and/or derivatives herein, can be engineered to express or overexpress one or more acyl-ACP reductases polypeptides, variants, or fragments thereof to achieve an improved production of one or more desirable branched fatty alcohols or derivatives. Alternatively, a host cell can be engineered to express an attenuated level of an endogenous acyl-ACP reductase. Non-limiting examples of suitable acyl-ACP reductases are listed in Table 7 below:







TABLE 7







Acyl-ACP Reductase Polypeptides








Organism
Accession No.





Synechococcus elongatus PCC7942
Synpcc7942_1594 (YP_400611)


Synechocystis sp.
sll0209 (NP_442146)


Cyanothece sp. ATCC51142
cce_1430 (YP_001802846)


Prochlorococcus marinus
CCMP1986 PMM0533 (NP_892651)


subsp. pastoris str.


Gloeobacter violaceus
PCC7421 NP_96091 (gll3145)


Nostoc punctiforme
PCC73102 ZP_00108837



(Npun02004176)


Anabaena variabilis
ATCC29413 YP_323044



(Ava_2534)


Synechococcus elongatus
PCC6301 YP_170761 (syc0051_d)


Nostoc sp.
PCC 7120 alr5284 (NP_489324)


Prochlorococcus marinus
CCMP1986 PMM0533 (NP_892651)


subsp. pastoris str.









In certain embodiments, a host cell, which is used to produce fatty alcohols and/or derivatives herein, can be further engineered to express or overexpress one or more fatty alcohol biosynthesis polypeptides, variants, or fragments thereof in order to achieve an improved production of one or more desirable branched fatty alcohols or derivatives. Alternatively, a host cell can be engineered to express an attenuated level of an endogenous fatty alcohol biosynthesis polypeptide. Non limiting examples of suitable fatty alcohol biosynthesis polypeptides are listed in Table 8 below:







TABLE 8







Fatty Alcohol Biosynthesis/Alcohol Dehydrogenase Polypeptide













GenBank







Accession

GenBank

GenBank


Name
No.
Name
Accession No.
Name
Accession No.





ygjB
NP_418690
YggP
YP_026187
YciK
NP_415787


yahK
NP_414859
YiaY
YP_026233
YgfF
NP_417378


adhP
NP_415995
FucO
NP_417279
YghA
NP_417476


ydjL
NP_416290
EutG
NP_416948
YjgI
NP_418670


ydjJ
NP_416288
YqhD
NP_417484
YdfG
NP_416057


idnD
NP_418688
AdhE
NP_415757
YgcW
NP_417254


Tdh
NP_418073
dkgB
NP_414743
UcpA
NP_416921


yjjN
NP_418778
YdjG
NP_416285
EntA
NP_415128


rspB
NP_416097
YeaE
NP_416295
FolM
NP_416123


gatD
NP_416594
dkgA
NP_417485
HdhA
NP_416136


yphC
NP_417040
YajO
NP_414953
HcaB
NP_417036


yhdH
NP_417719
YghZ
NP_417474
SrlD
NP_417185


ycjQ
NP_415829
Tas
NP_417311
KduD
NP_417319


yncB
NP_415966
YdhF
YP_025305
IdnO
NP_418687


Qor
NP_418475
YdbC
NP_415924
FabG
NP_415611


frmA
NP_414890
ybbO
NP_415026
FabI
NP_415804


ybdR
NP_415141
yohF
NP_416641
YdjA
NP_416279









In some instances, a host cell, which can be used to produce branched fatty alcohols and/or derivatives herein, is genetically engineered to increase the level of branched fatty acids in the host cell relative to a corresponding wild-type host cell. For example, the host cell can be genetically engineered to express a reduced level of an acyl-CoA synthase relative to a wild-type host cell. In one embodiment, the level of expression of one or more genes (e.g., an acyl-CoA synthase gene) is reduced by genetically engineering a “knock out” host cell. Any known acyl-CoA synthase gene can be reduced or knocked out in a host cell. Non-limiting examples of acyl-CoA synthase genes include fadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC—4074, fadDD35, fadDD22, faa3p or the gene encoding the protein ZP—01644857. Specific examples of acyl-CoA synthase genes include fadDD35 from M. tuberculosis H37Rv [NP—217021], fadDD22 from M. tuberculosis H37Rv [NP—217464], fadD from E. coli [NP—416319], fadK from E. coli [YP—416216], fadD from Acinetobacter sp. ADP1 [YP—045024], fadD from Haemophilus influenza RdkW20 [NP—438551], fadD from Rhodopseudomonas palustris Bis B18 [YP—533919], BH3101 from Bacillus halodurans C-125 [NP—243969], Pfl-4354 from Pseudomonas fluorescens Pfo-1 [YP—350082], EAV15023 from Comamonas testosterone KF-1 [ZP—01520072], yhfL from B. subtilis [NP—388908], fadD1 from P. aeruginosa PAO1 [NP—251989], fadD1 from Ralstonia solanacearum GM1 1000 [NP—520978], fadD2 from P. aeruginosa PAO1 [NP—251990], the gene encoding the protein ZP—01644857 from Stenotrophomonas maltophilia R551-3, faa3p from Saccharomyces cerevisiae [NP—012257], faa1p from Saccharomyces cerevisiae [NP—014962], lcfA from Bacillus subtilis [CAA99571], or those described in Shockey et al., Plant. Physiol., 129: 1710-1722 (2002); Caviglia et al., J. Biol. Chem., 279: 1163-1169 (2004); Knoll et al., J. Biol. Chem., 269(23): 16348-56 (1994); Johnson et al., J. Biol. Chem., 269: 18037-18046 (1994); and Black et al., J. Biol. Chem. 267: 25513-25520 (1992).
Production of Branched Precursors
Branched fatty alcohols and derivatives can be produced from branched fatty aldehydes containing one or more branched points, using branched acyl-ACPs as substrates for a fatty aldehyde biosynthesis polypeptide or an acyl-ACP reductase polypeptide as described herein. The first step in forming branched fatty alcohol precursors is the production of the corresponding alpha-keto acids by a branched-chain amino acid aminotransferase. Host cells may endogenously include genes encoding such enzymes or such genes can be recombinantly introduced. E. coli, for example, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42; GenBank accession YP—026247). In host cells where no branched-chain amino acid aminotransferase are expressed, an E. coli IlvE or any other branched-chain amino acid aminotransferase (e.g., IlvE from Lactococcus lactic (GenBank accession AAF34406), IlvE from Pseudomonas putida (GenBank accession NP—745648), or IlvE from Streptomyces coelicolor (GenBank accession NP—629657)), can be introduced.
In another embodiment, the production of alpha-keto acids can be achieved using the methods described in Park et al., PNAS, 104:7797-7802 (2007) and Atsumi et al., Nature, 451: 86-89 (2008). For example, 2-ketoisovalerate can be produced by overexpressing the genes encoding IlvI, IlvH, IlvH mutant, IlvB, IlvN, IlvGM, IlvC, or IlvD. Alternatively, 2-keto-3-methyl-valerate can be produced by overexpressing the genes encoding IlvA and IlvI, IlvH (or AlsS of Bacillus subtilis), IlvC, IlvD, or their homologs. 2-keto-4-methyl-pentanoate can also be produced by overexpressing the genes encoding IlvI, IlvH, IlvC, IlvD and LeuA, LeuB, LeuC, LeuD, or their homologs.
In another example, isobutyryl-CoA can be made in a host cell, for example in E. coli, through the coexpression of a crotonyl-CoA reductase (Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds, J. Bacteriol., 179: 5157 (1997)). Crotonyl-CoA is an intermediate in fatty acid biosynthesis in E. coli and other microorganisms. Non-limiting examples of ccr and icm genes from selected microorganisms are listed in Table 9.







TABLE 9







ccr and icm Genes from Selected Microorganisms











Organism
Gene
GenBank Accession #







Streptomyces coelicolor
Ccr
NP_630556




icmA
NP_629554




icmB
NP_630904



Streptomyces cinnamonensis
Ccr
AAD53915




icmA
AAC08713




icmB
AJ246005










Formation of Branched Cyclic Fatty Alcohols and Derivatives

Branched cyclic fatty alcohols can be produced from suitable alpha keto acids using branched cyclic fatty acid derivatives such as a branched cyclic acyl-ACP as substrates. To produce branched cyclic fatty acid derivative substrates, genes that provide cyclic precursors (e.g., the ans, chc, and plm gene families) can be introduced into a host cell and expressed to allow initiation of fatty acid biosynthesis from branched cyclic precursors. For example, to convert a host cell, such as E. coli, into one capable of synthesizing co-cyclic fatty acids (cyFA), a gene that provides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (Cropp et al., Nature Biotech., 18: 980-983 (2000)) can be introduced and expressed in the host cell. Non-limiting examples of genes that provide CHC-CoA in E. coli include: ansJ, ansK, ansL, chcA, and ansM from the ansatrienin gene cluster of Streptomyces collinus (Chen et al., Eur. J. Biochem., 261: 98-107 (1999)) or plmJ, plmK, plmL, chcA, and plmM from the phoslactomycin B gene cluster of Streptomyces sp. HK803 (Palaniappan et al., J. Biol. Chem., 278: 35552-35557 (2003)) together with the chcB gene (Patton et al., Biochem., 39: 7595-7604 (2000)) from S. collinus, S. avermitilis, or S. coelicolor (see Table 10). The genes listed in Table 10 can then be expressed to allow initiation and elongation of co-cyclic fatty acids. Alternatively, the homologous genes can be isolated from microorganisms that make cyFA and expressed in a host cell (e.g., E. coli).







TABLE 10







Genes for the Synthesis of CHC-CoA













GenBank



Organism
Gene
Accession No.







Streptomyces collinus
ansJK
U72144*




ansL




chcA




ansM




chcB
AF268489



Streptomyces sp. HK803
pmlJK
AAQ84158




pmlL
AAQ84159




chcA
AAQ84160




pmlM
AAQ84161



Streptomyces coelicolor
chcB/caiD
NP_629292



Streptomyces avermitilis
chcB/caiD
NP_629292







*Only chcA is annotated in GenBank entry U72144; ansJKLM are according to Chen et al. (Eur. J. Biochem., 261: 98-107 (1999)).






Genes fabH, acp, and fabF allow initiation and elongation of co-cyclic fatty acids because they have broad substrate specificity. If the coexpression of any of these genes with the genes listed in Table 10 does not yield cyFA, then fabH, acp, and/or fabF homologs from microorganisms that make cyFAs (e.g., those listed in Table 11) can be isolated (e.g., by using degenerate PCR primers or heterologous DNA sequence probes) and coexpressed.







TABLE 11







Non-Limiting Examples of Microorganisms Containing ω-cyclic


Fatty Acids








Organism
Reference





Curtobacterium pusillum
ATCC19096


Alicyclobacillus acidoterrestris
ATCC49025


Alicyclobacillus acidocaldarius
ATCC27009


Alicyclobacillus
Moore, J Org. Chem., 62: 2173 (1997)


cycloheptanicus*





*Uses cycloheptylcarbonyl-CoA and not cyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.






Branched Fatty Alcohol Saturation Levels

The degree of saturation in branched fatty acid derivative substrates, such as, for example, a branched acyl-ACP, (which can then be converted into branched fatty aldehydes and then branched fatty alcohols as described herein) can be controlled by regulating the degree of saturation of fatty acid intermediates. For example, the sfa, gns, and fab families of genes can be expressed or overexpressed to control the saturation of a branched acyl-ACP. In certain embodiments, the host cells can be engineered to reduce the expression of an sfa, gns, or fab gene and control the level of saturated substrates vs. unsaturated substrates, which in turn affects the production level of saturated branched fatty alcohols or derivatives vs. unsaturated branched fatty alcohols or derivatives.
In some instances, a host cell can be engineered to express an attenuated level of a dehydratase/isomerase and/or a ketoacyl-ACP synthase. For example, a host cell can be engineered to express a decreased level of fabA and/or fabB. In some instances, the host cell can be cultured or grown in the presence of unsaturated fatty acids. In some instances, the host cell can be engineered to express or overexpress a gene encoding a desaturases enzyme. One non-limiting example of a desaturases is B. subtilis DesA (AF037430). Other genes encoding desaturases are known in the art can be introduced or used in the host cell and methods described herein, such as desaturases that use acyl-ACPs, including, for example, hexadecanoyl-ACP or octadecanoyl-ACP.
In some embodiments, those cells can be engineered to produce unsaturated fatty acids by engineering the production host to overexpress fabB or by growing the production host at low temperatures (e.g., less than 37° C.). FabB has preference to cis-δ3decenoyl-ACP and results in unsaturated fatty acid production in E. coli. Overexpression of fabB results in the production of a significant percentage of unsaturated fatty acids (de Mendoza et al., J. Biol. Chem., 258: 2098-2101 (1983)). The gene fabB may be inserted into and expressed in host cells not naturally having the gene. These unsaturated fatty acids can then be used as intermediates in host cells that are engineered to produce branched and unsaturated fatty acid derivative substrates, such as branched and unsaturated fatty aldehydes, which can in turn be converted into branched and unsaturated fatty alcohols and derivatives.
In other instances, a repressor of fatty acid biosynthesis, for example, fabB (GenBank accession NP—418398), can be deleted, which will also result in increased unsaturated fatty acid production in E. coli (Zhang et al., J. Biol. Chem., 277: 15558 (2002)). Similar deletions may be made in other host cells. A further increase in unsaturated fatty acids may be achieved, for example, by overexpressing fabM (trans-2, cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) and controlled expression of fabK (trans-2-enoyl-ACP reductase II, GenBank accession NP—357969) from Streptococcus pneumoniae (Marrakchi et al., J. Biol. Chem., 277: 44809 (2002)), while deleting E. coli fabI (trans-2-enoyl-ACP reductase, GenBank accession NP—415804). In some examples, the endogenous fabF gene can be attenuated, thus increasing the percentage of palmitoleate (C16:1) produced.
Production of Genetic Variants
Variants can be naturally occurring or created in vitro. In particular, such variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.
Methods of making variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
For example, variants can be created using error prone PCR (see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCR Methods Applic., 2: 28-33 (1992)). In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Briefly, in such procedures, nucleic acids to be mutagenized (e.g., a fatty aldehyde biosynthetic polynucleotide sequence) are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized (e.g., a fatty aldehyde biosynthetic polynucleotide sequence), 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mM MgCl2, 0.5 mM MnCl2, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciated that these parameters can be varied as appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.
Variants can also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science, 241: 53-57 (1988).
Variants can also be generated by assembly PCR, which involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Pat. No. 5,965,408.
Still another method of generating variants is sexual PCR mutagenesis, wherein forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequence in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described in, for example, Stemmer, Proc. Natl. Acad. Sci. USA, 91: 10747-10751 (1994).
Variants can also be created by in vivo mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such “mutator” strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence (e.g., a BKD polynucleotide sequence, a beta acyl-ACP synthase polynucleotide sequence, a fatty aldehyde biosynthesis polynucleotide sequence, or a fatty alcohol biosynthesis polynucleotide sequence) in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described in, for example, International Publication WO 91/016427.
Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence. The oligonucleotide often contains a completely and/or partially randomized native sequence.
Recursive ensemble mutagenesis can also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, Arkin et al., Proc. Natl. Acad. Sci. USA, 89: 7811-7815 (1992).
In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res., 11: 1548-1552 (1993). Random and site-directed mutagenesis are described in, for example, Arnold, Curr. Opin. Biotech., 4: 450-455 (1993).
In some embodiments, variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.
Polynucleotide variants also include nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-halo, 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. (See, e.g., Summerton et al., Antisense Nucleic Acid Drug Dev., 7: 187-195 (1997); and Hyrup et al., Bioorgan. Med. Chem., 4: 5-23 (1996)). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
Production of Polypeptide Variants
Conservative substitutions are those that substitute an amino acid in a polypeptide by another amino acid of similar characteristics. Common conservative substitutions include, without limitation: replacing an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacing a serine with a threonine or vice versa; replacing an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacing a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; replacing a basic residue, such as lysine and arginine, with another basic residue; and replacing an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.
Other polypeptide variants are those in which one or more amino acid residues include a substituent group. Still other polypeptide variants are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol).
Additional polypeptide variants are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence, or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.
In some instances, the polypeptide variants described herein retain the same biological function as a polypeptide from which they are derived (e.g., retain branched-chain alpha keto acid dehydrogenase activity, retain beta ketoyacyl ACP synthase activity, such as FabH activity, or retain fatty aldehyde biosynthetic activity, such as carboxylic acid or fatty acid reductase activity) and have amino acid sequences substantially identical thereto.
In other instances, the polypeptide variants have 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%, at least about 95%, or more than about 95% homology to an amino acid sequence from which they are derived. In another embodiment, the polypeptide variants include a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
The polypeptide variants or fragments thereof can be obtained by isolating nucleic acids encoding them using techniques described herein or by expressing synthetic nucleic acids encoding them. Alternatively, polypeptide variants or fragments thereof can be obtained through biochemical enrichment or purification procedures. The sequence of polypeptide variants or fragments can be determined by proteolytic digestion, gel electrophoresis, and/or microsequencing. The sequence of the polypeptide variants or fragments can then be compared to the amino acid sequence from which it is derived using any of the programs described herein.
The polypeptide variants and fragments thereof can be assayed for enzymatic activity. For example, the polypeptide variants or fragments can be contacted with a substrate under conditions that allow the polypeptide variants or fragments to function. A decrease in the level of the substrate or an increase in the level of the desired product can be measured to determine its activity.
Modifications to Increase Conversion of Branched Substrates to Branched Fatty Alcohol
Host cells can be engineered using known polypeptides to produce branched fatty alcohols from branched substrate, including, for example, a branched fatty acid, a branched fatty acid derivative, a branched acyl-CoA, or a branched acyl-CoA derivative substrate. For example, one method of making branched fatty alcohols involves increasing the expression of, or expressing more active forms of fatty alcohol forming acyl-CoA reductases (encode by a gene such as acr1 from FAR, EC 1.2.1.50/1.1.1) or acyl-CoA reductases (EC 1.2.1.50) and alcohol dehydrogenase (EC 1.1.1.1).
The host cell can also be, for example, modified or engineered, such that it expresses or overexpresses at least one (E1) subunit of a branched-chain alpha keto acid dehydrogenase complex, and a beta ketoacyl-ACP synthase. The host cell can be further engineered such that it expresses or overexpresses a fatty aldehyde biosynthesis polypeptide and/or a fatty alcohol biosynthesis polypeptide. Alternatively, the host cell can be engineered such that it expresses or overexpresses an acyl-ACP reductase polypeptide and a fatty alcohol biosynthesis polypeptide.
In certain embodiments, the gene encoding the subunits of branched-chain alpha keto acid dehydrogenase complex can be derived from a bacterium, a plant, an insect, a yeast, a fungus, or a mammal. For example, the subunits of the branched-chain alpha keto acid dehydrogenase complex can be derived from a bacterium that uses branched amino acids as carbon source, including, for example, Pseudomonas putida or Bacillus subtilis. In another example, the branched-chain alpha-keto acid dehydrogenase complex polypeptide can be from a bacterium that comprises branched fatty acids in its phospholipids, including, e.g., a Legionella, Stenotrophomonas, Alteromonas, Flavobacterium, Myxococcus, Bccteroides, Micrococcus, Staphylococcus, Bacillus, Clostridium, Listeria, Lactococcus, or Streptomyces. In some embodiments, the bacterium is a Leginella pneumophila, Stenotrophomonas maltophilia, Alteromonas macleodii, Flabobacterium phsychrophilum, Myxococcus Xanthus, Bacteroides thetaiotaomicron, Macrococcus luteus, Staphylococcus aureus, Clostridium thermocellum, Listeria monocytogenes, Streptomyces lividans, Streptomyces coelicolor, Streptomyces glaucescens, Streptococcus pneumoniae, Streptomyces peucetius, Streptococcus pyogenes, Escherichia coli, Escherichia coli K-12, Lactococcus lactis ssp. Lactis, Mycobacterium tuberculosis, Enterococcus tuberculosis, Bacillus subtilis, Lactobacillus plantarum. In some embodiments, suitable fatty aldehyde biosynthesis polypeptides, fatty alcohol biosynthesis polypeptides, acyl-ACP reductases, and other polypeptides of the invention can be from a mycobacterium selected from Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium marinum, or Mycobacterium ulcerans. In other embodiments, the bacterium is Nocardia sp. NRRL 5646, Nocardia farcinica, Streptomyces griseus, Salinispora arenicola, or Clavibacter michiganenesis. In yet further embodiments, the polypeptide of the invention is derived from a cyanobacterium, including, for example, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, Cyanothece sp. ATCC51142, Prochlorococcus marinus subsp. pastoris str. CCMP1986 PMM0533, Gloeobacter violaceus PCC7421, Nostoc punctiforme PCC73102, Anabaena variabilis ATCC29413, Synechococcus elongatus PCC6301, and Nostoc sp. PCC 7120, Microcoleus chthonoplastes PCC7420, Arthrospira maxima CS-328, Lyngbya sp. PCC8106, Nodularia spumigena CCY9414, Trichodesmium erythraeum IMS101, Microcystis aeruginosa, Nostoc azollae, Anabaena variabilis, Crocophaera watsonii, Thermosynechococcus elongatus, Gloeobacer violaceus, Cyanobium, or Prochlorococcus marinus. 
Genetic Engineering of Host Cells to Produce Branched Fatty Alcohols
Various host cells can be used to produce branched fatty alcohols, as described herein. A host cell can be any prokaryotic or eukaryotic cell. For example, the host cell can be bacterial cells (such as E. coli), insect cells, yeast, or mammalian cells (such as Chinese hamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplary host cells include cells from the members of the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia, or Streptomyces. Yet other exemplary host cells can be a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus licheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, a Bacillus amyloliquefaciens cell, a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhizomucor miehei cell, a Mucor michei cell, a Streptomyces lividans cell, a Streptomyces murinus cell, or an Actinomycetes cell. Host cells can also be cyanobacterial cells such as, for example, Synechoccus sp., Synechoccus elongatus, or Synechocystis sp. cells.
In a preferred embodiment, the host cell is an E. coli cell, a Saccharomyces cerevisiae cell, or a Bacillus subtilis cell. For example, the host cell can be one from E. coli strain B, C, K, or W. Other suitable host cells are known to those skilled in the art.
Various methods well known in the art can be used to genetically engineer host cells to produce branched fatty alcohols. The methods can include the use of vectors, preferably expression vectors, containing a nucleic acid encoding the first (E1 alpha/beta) subunit of a branched-chain alpha keto acid dehydrogenase, and optionally also the second (E2) and/or the third (E3) subunit of that enzyme, and/or a beta ketoacyl-ACP synthase, and/or a fatty aldehyde biosynthetic polypeptide, and/or an alcohol dehydrogenase, and/or an acyl-ACP reductases, described herein, polypeptide variant, or a fragment thereof. Those skilled in the art will appreciate a variety of viral vectors (for example, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors) and non-viral vectors can be used in the methods described herein.
The recombinant expression vectors can include polynucleotides described herein in a form suitable for expression in a host cell. The recombinant expression vectors can include one or more control sequences, selected on the basis of the host cell to be used for expression. The control sequence is operably linked to the nucleic acid sequence to be expressed. Such control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the nucleic acids as described herein.
In some embodiments, recombinant expression vectors can be designed for expression of a gene encoding a first (E1 alpha/beta) subunit, and optionally a second (E2) and/or a third (E3) subunit of a branched-chain alpha-keto acid dehydrogenase (or variant) and/or a gene encoding a beta-ketoacyl ACP synthase (or variant), and/or a gene encoding a fatty aldehyde biosynthesis polypeptide (or variant), and/or a gene encoding an alcohol dehydrogenase (or variant), and/or a gene encoding an acyl-ACP reductases (or variant) in a suitable host cell. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, by using T7 promoter regulatory sequences and T7 polymerase.
Expression of genes encoding polypeptides in prokaryotes, for example, E. coli, is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.
Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), pp. 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident X, prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize expression is to express the polypeptide in a host cell with an impaired capacity to proteolytically cleave the recombinant polypeptide (see Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), pp. 119-128). Another strategy is to alter the nucleic acid sequence to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the host cell (Wada et al., Nucleic Acids Res., 20: 2111-18 (1992)). These strategies can be carried out by standard DNA synthesis techniques.
In another embodiment, the host cell is a yeast cell, and the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).
Alternatively, polypeptides described herein can be expressed in insect cells using baculovirus expression vectors. Available baculovirus vectors include, for example, the pAc series (Smith et al., Mol. Cell. Biol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)).
In yet another embodiment, the polypeptides described herein can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature, 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBO J., 6: 187-195 (1987)). When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. Commonly used promoters include those derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. Other suitable expression systems for both prokaryotic and eukaryotic cells are described in chapters 16-17 of Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
It is known that, depending upon the expression vector and transformation technique used, only a small fraction of bacterial cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs, such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
It is known that, depending upon the expression vector and transfection technique used, only a small fraction of mammalian cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
Transport Proteins
Transport proteins can export or excrete polypeptides and organic compounds (e.g., branched fatty alcohols) out of a host cell. A number of transport and efflux proteins can be modified to selectively secrete particular types of compounds such as branched fatty alcohols.
Non-limiting examples of suitable transport proteins are ATP-Binding Cassette (ABC) transport proteins, efflux proteins, and fatty acid transporter proteins (FATP). Additional suitable transport proteins include the ABC transport proteins from organisms such as Caenorhabditis elegans, Arabidopsis thalania, Alkaligenes eutrophus, or Rhodococcus erythropolis. Exemplary ABC transport proteins include, without limitation, CER5, AtMRP5, AmiS2, and AtPGP1. Host cells can also be chosen for their endogenous ability to secrete organic compounds. The efficiency of organic compound production and secretion into the host cell environment (e.g., culture medium, fermentation broth) can be expressed as a ratio of intracellular product to extracellular product. For example, the ratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.
Fermentation
The production and isolation of branched fatty alcohols can be enhanced by employing beneficial fermentation techniques. One method for maximizing production while reducing costs is increasing the percentage of the carbon source that is converted to the branched fatty alcohol products.
During normal cellular lifecycles, carbon is used in cellular functions such as producing lipids, saccharides, proteins, organic acids, and nucleic acids. Reducing the amount of carbon necessary for growth-related activities can increase the efficiency of carbon source conversion to product. This can be achieved by, for example, first growing host cells to a desired density (for example, a density achieved at the peak of the log phase of growth). At such a point, replication checkpoint genes can be harnessed to stop the growth of cells. Specifically, quorum sensing mechanisms (reviewed in Camilli et al., Science, 311: 1113 (2006); Venturi FEMS Microbio. Rev., 30: 274-291 (2006); and Reading et al., FEMS Microbiol. Lett., 254: 1-11 (2006)) can be used to activate checkpoint genes, such as p53, p21, or other checkpoint genes.
Genes that can be activated to stop cell replication and/or growth in E. coli include umuDC genes. The overexpression of umuDC genes stops the progression from stationary phase to exponential growth (Murli et al., J. Bact., 182: 1127 (2000)). UmuC is a DNA polymerase that can carry out translesion synthesis over non-coding lesions—the mechanistic basis of most UV and chemical mutagenesis. The umuDC gene products are involved in the process of translesion synthesis and also serve as a DNA sequence damage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′, UmuD′2C, UmuD′2, and UmuD2. Simultaneously, product-producing genes can be activated, thus minimizing the need for replication and maintenance pathways to be used while a fatty aldehyde is being made. Host cells can also be engineered to express umuC and umuD from E. coli in pBAD24 under the prpBCDE promoter system through de novo synthesis of this gene with the appropriate end-product production genes.
The percentage of input carbons converted to branched fatty alcohols can be a cost driver. The more efficient the process is (i.e., the higher the percentage of input carbons converted to branched fatty alcohols), the less expensive the process will be. For oxygen-containing carbon sources (e.g., glucose and other carbohydrate based sources), the oxygen must be released in the form of carbon dioxide. For every 2 oxygen atoms released, a carbon atom is also released leading to a maximal theoretical metabolic efficiency of approximately 34% (w/w) (for fatty acid derived products). This figure, however, changes for other organic compounds and carbon sources. Typical efficiencies in the literature are approximately less than 5%. Host cells engineered to produce fatty alcohols can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, host cells can exhibit an efficiency of about 10% to about 25%. In other examples, such host cells can exhibit an efficiency of about 25% to about 30%. In other examples, host cells can exhibit greater than 30% efficiency.
The host cell can be additionally engineered to express recombinant cellulosomes, such as those described in International Publication WO 2008/100251. These cellulosomes can allow the host cell to use cellulosic material as a carbon source. For example, the host cell can be additionally engineered to express invertases (EC 3.2.1.26) so that sucrose can be used as a carbon source. Similarly, the host cell can be engineered using the teachings described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; and 5,602,030, so that the host cell can assimilate carbon efficiently and use cellulosic materials as carbon sources.
In one example, the fermentation chamber can enclose a fermentation that is undergoing a continuous reduction. In this instance, a stable reductive environment can be created. The electron balance can be maintained by the release of carbon dioxide (in gaseous form). Efforts to augment the NAD/H and NADP/H balance can also facilitate in stabilizing the electron balance. The availability of intracellular NADPH can also be enhanced by engineering the host cell to express an NADH:NADPH transhydrogenase. The expression of one or more NADH:NADPH transhydrogenases converts the NADH produced in glycolysis to NADPH, which can enhance the production of fatty alcohols.
For small scale production, the engineered host cells can be (a) grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L, (b) fermented, and (c) induced to express desired bkd genes, beta-ketoacyl ACP synthase genes, fatty aldehyde biosynthesis genes, alcohol dehydrogenase genes, and/or acyl-ACP reductases genes, based on the specific genes encoded in the appropriate plasmids. For large scale production, the engineered host cells can be (a) grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, 1,000,000 L, or larger, (b) fermented, and (c) induced to express the desired bkd genes, beta-ketoacyl ACP synthase genes, fatty aldehyde biosynthesis genes, alcohol dehydrogenase genes, and/or acyl-ACP reductases genes based on the specific genes encoded in the plasmids or incorporated into the host cell's genome.
For example, a suitable production host, such as an E. coli, harboring plasmids containing the desired genes or having the genes integrated in its chromosome can be incubated in a suitable reactor, for example a 1 L reactor, for 20 hours at 37° C. in an M9 medium supplemented with 2% glucose, carbenicillin, and chloramphenicol. When the OD600 of the culture reaches 0.9, the production host can be induced with IPTG alcohol After incubation, the spent media can be extracted and the organic phase can be examined for the presence of branched fatty alcohols using GC-MS.
In some instances, after the first hour of induction, aliquots of no more than about 10% of the total cell volume can be removed each hour and allowed to sit without agitation to allow the branched fatty alcohols to rise to the surface and undergo a spontaneous phase separation or precipitation. The branched fatty alcohol component can then be collected, and the aqueous phase returned to the reaction chamber. The reaction chamber can be operated continuously. When the OD600 drops below 0.6, the cells can be replaced with a new batch grown from a seed culture.
Producing Branched Fatty Alcohols and Derivatives Using Cell-Free Methods
In some embodiments, branched fatty alcohols can be produced using a purified polypeptide (e.g., a branched-chain alpha keto acid dehydrogenase complex polypeptide) described herein and a substrate (e.g., an alpha keto acid, malonyl-CoA, 2-oxo-isovalerate, 2-oxo-isobutylrate, 2-oxo-3-methyl-valerate. 2-oxo-isocaproate, 2-oxoglutarate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate, or pyruvate) produced, for example, by a method described herein. For example, a host cell can be engineered to express a branched-chain alpha keto acid dehydrogenase polypeptide or the E1 (alpha and beta), and optionally, the E2 and/or the E3 subunits thereof, or variants as described herein. The host cell can be cultured under conditions sufficient to allow expression of the polypeptide. Cell free extracts can then be generated using known methods, including, for example, cell lysis using detergents or sonication. The expressed polypeptides can be purified. Thereafter, substrates described herein can be added to the cell free extracts and maintained under conditions to allow conversion of the substrates (e.g., alpha keto acids, such as 2-oxo-isovalerate, 2-oxo-isobutylrate, 2-oxo-3-methyl-valerate. 2-oxo-isocaproate, 2-oxoglutarate, 2-oxopentanoate, 3-methyl-2-oxobutanoate, 3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate, or pyruvate) to branched chain acyl-CoAs, which can then be converted into branched fatty aldehydes and branched fatty alcohols. The branched fatty alcohols can then be separated and purified using known techniques.
Post-Production Processing
Depending on the intended use of the branched fatty alcohols produced in accordance with the methods here, post-production processing may or may not be necessary. As such, in certain industrial applications, the produced branched fatty alcohols and/or derivatives may be suitably used per se as surfactants. Moreover, such surfactants can be directly blended or formulated into suitable cleaning compositions.
The branched fatty alcohols produced during fermentation can be separated from the fermentation media, using any known technique for separating fatty alcohols from aqueous media. One exemplary separation process is a two phase (bi-phasic) separation process, which involves fermenting the genetically engineered host cells under conditions sufficient to produce a branched fatty alcohol, allowing it to collect in an organic phase, and separating the organic phase from the aqueous fermentation broth. This method can be practiced in both a batch and continuous fermentation processes.
Bi-phasic separation uses the relative immiscibility of fatty alcohols to facilitate separation. Immiscible refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient. One of ordinary skill in the art will appreciate that by choosing a fermentation broth and organic phase, such that the branched fatty alcohol being produced has a high log P value, the branched fatty alcohol can separate into the organic phase, even at very low concentrations, in the fermentation vessel.
The branched fatty alcohols produced by the methods described herein can be relatively immiscible in the fermentation broth and the cytoplasm. Therefore, the branched fatty alcohol can collect in an organic phase either intracellularly or extracellularly. The collection of the products in the organic phase can lessen the impact of the branched fatty alcohol on cellular function and can allow the host cell to produce more product.
The branched fatty alcohol can thus be produced as a homogeneous compounds wherein at least about 60%, 70%, 80%, 90%, or 95% of the branched fatty alcohols produced will have carbon chain lengths that vary by less than about 6 carbons, less than about 4 carbons, or less than about 2 carbons. These compounds can also be produced with a relatively uniform degree of saturation. They can be used per se as surfactants or can be formulated into suitable cleaning compositions. They can also be used as fuels, fuel additives, starting materials for production of other chemical compounds (e.g., polymers, surfactants, plastics, textiles, solvents, adhesives, etc.), or personal care additives. These compounds can also be used as feedstock for subsequent reactions, for example, hydrogenation, catalytic cracking (e.g., via hydrogenation, pyrolysis, or both), and can be dehydrated to make other products. In particular, these branched products confer low volatility, beneficial low-temperature properties, as well as oxidative stability, making them ideal for low temperature applications such as in household cleaning compositions and personal and beauty care products.
In some embodiments, the branched fatty alcohols produced using methods described herein can contain between about 50% and about 90% carbon, or between about 5% and about 25% hydrogen. In other embodiments, the branched fatty alcohols produced using methods described herein can contain between about 65% and about 85% carbon, or between about 10% and about 15% hydrogen.
In some embodiments, the branched fatty alcohols produced in accordance with the disclosure herein comprises a C6-C26 branched fatty alcohol. In some embodiments, the branched fatty alcohol comprises a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 branched fatty alcohol. In particular embodiments, the branched fatty alcohol is a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 branched fatty alcohol. In certain embodiments, the hydroxyl group of the branched fatty alcohol is in the primary (C1) position. In certain embodiment, the branched fatty alcohol is an iso-fatty alcohol or an anteiso-fatty alcohol. In exemplary embodiments, the branched fatty alcohol is selected from iso-C7:0, iso-C8:0, iso-C10:0, iso-C11:0, iso-C12:0, iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0, iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0, anteiso-C11:0, anteiso-C12:0, anteiso-C13:0, anteiso-C14:0, anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and anteiso-C19:0 fatty alcohol.
In certain embodiments, the fatty alcohol product can comprise straight chain fatty alcohols. In other embodiments, the branched fatty alcohols produced by the host cells described herein can comprise one or more points of branching. In certain embodiments, the branched fatty alcohols produced by the host cells as described herein can comprise one or more cyclic moieties.
In some embodiments, the branched fatty alcohols can be unsaturated branched fatty alcohols. For example, the branched fatty alcohols produced in accordance with the present description can be monounsaturated branched fatty alcohols. In certain embodiments, the unsaturated branched fatty alcohol can be a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated branched fatty alcohol. In other embodiments, the branched fatty alcohol is unsaturated at the omega-7 position. In certain embodiments, the unsaturated branched fatty alcohol comprises a cis double bond.
In some embodiments, branched fatty alcohols are produced at a relative yield to a straight-chain fatty alcohol at about 20%, for example, at about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or higher. In an exemplary embodiment, the total amount of branched fatty alcohols produced is estimated to about 45% to about 50% relative to the amount of straight-chain fatty alcohols produced by a host cell.
In any of the aspects described herein, the production yield of fatty alcohols, including branched fatty alcohols and straight chain fatty alcohol, is about 1 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850 mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950 mg/L, about 975 mg/L, about 1000 g/L, about 1050 mg/L, about 1075 mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175 mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275 mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375 mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475 mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575 mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675 mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775 mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875 mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975 mg/L, about 2000 mg/L, or more.
In another aspect, the branched fatty alcohol produced in accordance with the present invention is produced by culturing a host cell described herein in a medium having a low level of iron, under conditions sufficient to produce a branched fatty alcohol. In particular embodiments, the medium contains less than about 500 μM iron, less than about 400 μM iron, less than about 300 μM iron, less than about 200 μM iron, less than about 150 μM iron, less than about 100 μM iron, less than about 90 μM iron, less than about 80 μM iron, less than about 70 μM iron, less than about 60 μM iron, less than about 50 μM iron, less than about 40 μM iron, less than about 30 μM iron, less than about 20 μM iron, less than about 10 μM iron, or less than about 5 μM iron. In certain embodiments, the medium does not contain iron.
Bioproducts (e.g., surfactants and cleaning compositions) comprising microbially produced branched fatty alcohols and/or derivatives, produced using the fatty acid biosynthetic pathway, have not been produced from renewable sources and, as such, are new compositions of matter. These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14C dating. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference).
The ability to distinguish bioproducts from petroleum based organic compounds is beneficial in tracking these materials in commerce. Organic compounds or chemicals comprising both biologically based and petroleum based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum based materials. Hence, the surfactants and cleaning compositions of the present invention be followed in commerce on the basis of their unique carbon isotope profile.
Surfactants or cleaning compositions produced in accordance with the present disclosure can be distinguished from petroleum-derived compounds by comparing the stable carbon isotope ratio (13C/12C) of each. The 13C/12C ratio in a given bioproduct is a consequence of the 13C/12C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed. It also reflects the precise metabolic pathway. Regional variations also occur. Petroleum, C3 plants (the broadleaf), C4 plants (the grasses), and marine carbonates all show significant differences in 13C/12C and the corresponding δ13C values. Moreover, lipid matter of C3 and C4 plants analyze differently than materials derived from the carbohydrate components of the same plants as a consequence of the metabolic pathway.
Within the precision of measurement, 13C shows large variations due to isotopic fractionation effects, the most significant of which for bioproducts is the photosynthetic mechanism. The major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO2). Two large classes of vegetation are those that incorporate the “C3” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C4” (or Hatch-Slack) photosynthetic cycle.
In C3 plants, the primary CO2 fixation/carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound. C3 plants, such as hardwoods and conifers, are dominant in the temperate climate zones.
In C4 plants, an additional carboxylation reaction involving phosphoenol-pyruvate carboxylase, is the primary carboxylation reaction. The first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated. The CO2 thus released is refixed by the C3 cycle. Examples of C4 plants are tropical grasses, corn, and sugar cane.
Both C4 and C3 plants exhibit a range of 13C/12C isotopic ratios, but typical values are about −7 to about −13 per mil for C4 plants and about −19 to about −27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon, 19: 355 (1977)). Coal and petroleum fall generally in this latter range. The 13C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material. The “δ13C” values are expressed in parts per thousand (per mil), abbreviated, ‰, and are calculated as follows:

δ13C(‰)=[(13C/12C)sample−(13C/12C)standard]/(13C/12C)standard×1000

Since the PDB reference material (RM) has been exhausted, a series of alternative RMs have been developed in cooperation with the IAEA, USGS, NIST, and other selected international isotope laboratories. Notations for the per mil deviations from PDB is δ13C. Measurements are made on CO2 by high precision stable ratio mass spectrometry (IRMS) on molecular ions of masses 44, 45, and 46.
The branched fatty alcohol and derivative compositions as well as the surfactants or cleaning compositions described herein include bioproducts produced by any of the methods described herein. The surfactants and cleaning compositions can have a δ13C of about −28 or greater, about −27 or greater, −20 or greater, −18 or greater, −15.4 or greater, −15 or greater, −13 or greater, −10 or greater, or −8 or greater. A surfactant or cleaning composition so produced can have a δ13C of about −30 to about −15, about −27 to about −19, about −25 to about −21, about −15 to about −5, about −15.4 to about −10.9, about −13.92 to about −13.84, about −13 to about −7, or about −13 to about −10. For example it can have a δ13C of about −10, −11, −12, or −12.3.
The surfactants or cleaning compositions herein can also be distinguished from petroleum-derived compounds by comparing the amount of 14C in each compound. Because 14C has a nuclear half life of 5730 years, petroleum based chemicals containing “older” carbon can be distinguished from bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles,” Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) (1992), pp. 3-74).
The basic assumption in radiocarbon dating is that the constancy of 14C concentration in the atmosphere leads to the constancy of 14C in living organisms. But because of atmospheric nuclear testing since 1950 and the burning of fossil fuel since 1850, 14C has acquired a second, geochemical time characteristic. Its concentration in atmospheric CO2, and hence in the living biosphere, approximately doubled at the peak of nuclear testing, in the mid-1960s. It has since been gradually returning to the steady-state cosmogenic (atmospheric) baseline isotope rate (14C/12C) of about 1.2×10−12, with an approximate relaxation “half-life” of 7-10 years. (This latter half-life is not to be taken literally; rather, the detailed atmospheric nuclear input/decay function to trace the variation of atmospheric and biospheric 14C since the onset of the nuclear age should be used).
It is this latter biospheric 14C time characteristic that holds out the promise of annual dating of recent biospheric carbon. 14C can be measured by accelerator mass spectrometry (AMS), with results given in units of “fraction of modern carbon” (fM). fM is defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C. As used herein, “fraction of modern carbon” or “fM” has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), fM is approximately 1.1.
The invention provides surfactants or cleaning compositions, having an fM 14C of at least about 1. An exemplary surfactant has an fM 14C of at least about 1.01, of at least about 1.5, an fM 14C of about 1 to about 1.5, an fM 14C of about 1.04 to about 1.18, or an fM 14C of about 1.111 to about 1.124. Likewise, an exemplary cleaning composition has an fM 14C of at least about 1.01, of at least about 1.5, an fM 14C of about 1 to about 1.5, an fM 14C of about 1.04 to about 1.18, or an fM 14C of about 1.111 to about 1.124.
Another measurement of 14C is known as the percent of modern carbon, pMC. For an archaeologist or geologist using 14C dates, AD 1950 equals “zero years old”. This also represents 100 pMC. “Bomb carbon” in the atmosphere reached almost twice the normal level in 1963 at the peak of thermonuclear weapons testing. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 107.5 pMC. This means that a fresh biomass material, such as corn, would give a 14C signature near 107.5 pMC. Petroleum based compounds will have a pMC value of zero. Combining fossil carbon with present day carbon will result in a dilution of the present day pMC content. By presuming 107.5 pMC represents the 14C content of present day biomass materials and 0 pMC represents the 14C content of petroleum based products, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 107.5 pMC. If that material was diluted 50% with petroleum based products, it would give a radiocarbon signature of approximately 54 pMC.
A biologically based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum based material.
A surfactant or a cleaning composition comprising branched fatty alcohols and/or derivatives described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, such a surfactant or cleaning composition can have a pMC of between about 50 and about 100; between about 60 and about 100; between about 70 and about 100; between about 80 and about 100; between about 85 and about 100; between about 87 and about 98; or between about 90 and about 95. In yet other instances, it can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
Accordingly the present invention is drawn to a branched fatty alcohol or a derivative thereof produced by an engineered microbial host cell. The engineered microbial host cell expresses: (a) a first gene encoding a first polypeptide having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15, or of a variant thereof; and (b) a second gene encoding a second polypeptide having at least about 85% sequence identity to the amino acid sequence of any one or SEQ ID NOs:24, 26, 28, 30, 32, 34, 36, and 38, or of a variant thereof, and is cultured in the presence of one or more biological substrates of the first and second polypeptides. In some embodiments, the microbial host cell is engineered to express a third gene encoding a third polypeptide comprising an amino acid sequence having at least an about 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs:47, 49, 51, 53, 55, 57, 59, and 61, or of a variant thereof. In some embodiments, the microbial host cell is engineered to express a fourth gene encoding a fourth polypeptide comprising an amino acid sequence having at least an about 85% sequence identity to the amino acid sequence of any one of SEQ ID NO:69, 71, 73, 75, 77, 79, 81, and 83, or of a variant thereof. In any of the above embodiments, the microbial host cell is engineered to express a beta-ketoacyl ACP synthase gene in the host cell, wherein the beta-ketoacyl ACP gene encodes a polypeptide comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs:90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, or of a variant thereof. The beta-ketoacyl ACP synthase is, for example, a FabH that has specificity for branched-chain acyl-CoA substrates. In any of the embodiments above, the microbial host cell is engineered to express a fatty aldehyde biosynthesis polypeptide, or a variant thereof. In any of the embodiments above, the microbial host cell is engineered to express an acyl-ACP reductase polypeptide or a variant thereof, to modify the expression of a gene encoding a fatty acid synthase, which comprise expressing a gene encoding a thioesterase in the microbial host cell, to express a gene encoding an alcohol dehydrogenase or a variant thereof, and/or to express an attenuated level of a fatty acid degradation enzyme relative to a wild type host cell. The fatty acid degradation enzyme is, for example, an acyl-CoA synthase.
Branched Fatty Alcohol Derivatives
A derivative of the branched fatty alcohol produced in accordance to the methods described herein can be produced by converting the isolated branched fatty alcohol into a branched fatty alcohol derivative thereof. The branched fatty alcohol derivative can be any suitable branched fatty alcohol derivative selected from, for example, a branched fatty ether sulfate, a branched fatty phosphate ester, an alkylbenzyldimethyl-ammonium chloride, a branched fatty amine oxide, a branched fatty alcohol sulfate, a branched alkyl polyglucoside, a branched alkyl glyceryl ether sulfonate, and a branched ethoxylated fatty alcohol. Typically, the branched fatty alcohol derivative comprises an alkyl group that is about 6 to about 26 carbons in length. Preferably, the branched fatty alcohol comprises an alkyl group that is about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons in length. In certain embodiments, the alkyl group comprises one or more points of branching. In this regard, the number of carbons in the alkyl group refers to the hydrocarbon group derived from the branched fatty alcohol, and not to any carbon atoms added in the preparation of the branched fatty alcohol derivative, such as polyethoxy groups and the like.
As used herein, the term “fatty ether sulfate” is the same as “alkyl ether sulfate” wherein the alkyl residue is a fatty residue, and denotes a compound of the structure: RO(CH2CH2O)n—SO3H, wherein R is a C6-C26 alkyl group as defined herein, and n is an integer of 1 to about 50. Fatty ether sulfate can also refer to the salt denoted by RO(CH2CH2O)nSO3X, where n and R are as defined above and X is a cation. An exemplary fatty ether sulfate salt is a sodium salt, for example, RO(CH2CH2O)nSO3Na. In an exemplary embodiment, the R group comprises one or more points of branching.
As used herein, the term “fatty alcohol sulfate” denotes a compound of the structure: ROSO3H wherein R is a C8-C26 alkyl group. Fatty alcohol sulfate can also refer to the salt of the above structure, denoted by ROSO3X where R is as defined above and X is a cation. An exemplary fatty alcohol sulfate salt is a sodium salt, for example, ROSO3Na. In an exemplary embodiment, the R group comprises one or more points of branching.
As used herein, the term “fatty phosphate ester” is the same as “alkyl phosphate ester” wherein the alkyl residue is a fatty residue, and denotes a compound of the structure:

ROP(O)(OH)2 

As used herein, alkylbenzyldimethylammonium chlorides have the structure:




wherein R is a C8-C26 alkyl group as defined herein. For example, the alkyl group of R comprises one or more points of branching.

As used herein, the term “fatty amine oxide” is the same as “alkyl amine oxide” wherein the alkyl residue is a fatty residue as defined herein, and denotes a compound of the structure:




wherein R is a C8-C26 alkyl group as defined herein and wherein R1 and R2 are C1-C26 alkyl groups, preferably C1-C6 alkyl groups. Preferably the alkyl groups of R, R1 and R2 each independently comprises one or more points of branching.

Alkyl polyglucosides have the structure: RO(CnH2nO)tZx wherein R is a C8-C26 alkyl group, preferably comprising one or more points of branching, Z is a glucose residue, n is 2 or 3, t is from 0 to 10, and x is from about 1 to 10, preferably from about 1.5 to 4.
Alkyl glyceryl ether sulfonates have the structure:




wherein R is a C8-C26 alkyl group as defined herein, preferably comprising one or more points of branching, and n is an integer from 1 to 4, for example, 1, 2, 3, or 4.

As used herein, the term “fatty alcohol alkoxylate” is the same as “alkoxylated fatty alcohol” and denotes a compound of the structure: RO(CH2CH2)nOH wherein R is a C8-C26 alkyl group as defined herein and n is an integer from 1 to about 50. Preferably R comprises one or more points of branching.
The branched fatty alcohol derivatives can be produced by any suitable method, many of which are known in the art. See, e.g., “Handbook on Soaps, Detergents, and Acid Slurry,” 2nd ed., NIIR Board, Asia Pacific Business Press, Inc., Delhi, India.
In one embodiment, the branched fatty alcohol derivative is an ethoxylated branched fatty alcohol, which is also known in the art as a branched fatty alcohol ethoxylate, and has a structure as described herein. Preferably, the ethoxylated branched fatty alcohol contains from about 1 to about 50 moles of ethylene oxide per mole of branched fatty alcohol.
Surfactants or Detersive Surfactants
A surfactant composition of the present invention can comprise about 0.001 wt. % to about 100 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof. Preferably, a surfactant composition is a blend of a microbially produced branched fatty alcohol and/or derivative in combination with one or more other surfactants and/or surfactant systems that have been derived from similar (e.g., microbially derived) or different sources (e.g., synthetic, petroleum-derived). Those other surfactants and/or surfactant systems can confer additional desirable properties. In some embodiments, the one or more other surfactants and/or surfactant systems that are blended with the microbially produced branched fatty alcohols and/or derivatives can comprise linear or branched fatty alcohol derivatives, or they can be other types of surfactants such as, cationic surfactants, anionic surfactants and/or amphoteric/zwitterionic surfactants. These other surfactants and/or surfactants systems are collectively referred to as “co-surfactants” herein. For example, a surfactant composition of the invention can be a blend of a microbially produced branched fatty alcohol and/or derivative composition prepared in accordance with the disclosure herein, and a cationic surfactant derived from a petrochemical source, and the resulting surfactant composition only has good cleaning properties but also contributes certain disinfecting and/sanitizing benefits.
The cleaning composition of the invention can comprise, in addition to the microbially produced branched fatty alcohols and/or derivatives, or the surfactants comprising such branched materials and/or derivatives, co-surfactants selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, squitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. When present, the total amount of surfactants, including the microbially produced branched fatty alcohols and/or derivatives thereof, and the co-surfactants, is typically present at a level of about 0.1 wt. % or higher (e.g., about 1.0 wt. % or higher, about 10 wt. % or higher, about 25 wt. % or higher, about 50 wt. % or higher, about 70 wt. % or higher). For example, the total amount of surfactant in a cleaning composition can vary from about 0.1 wt. % to about 80 wt. % (e.g., from about 0.1 wt. % to about 40 wt. %, from about 0.1 wt % to about 12 wt. %, from about 1.0 wt. % to about 50 wt. %, or from about 5 wt. % to about 40 wt. %).
Various known surfactants can be suitable co-surfactants. In some embodiments, the co-surfactant can comprise an anionic surfactant. In certain embodiments, the amount of one or more anionic surfactants in the cleaning composition can be, for example, about 1 wt. % or more (e.g., about 5 wt. % or more, about 10 wt. % or more, about 20 wt. % or more, about 30 wt. % or more, about 40 wt. % or more). For example, the amount of one or more anionic surfactants in the cleaning composition can vary from about 1 wt. % to about 40 wt. %. Suitable anionic surfactants include, for example, linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid or soap. In some embodiments, an anionic surfactant is, for example, a C10-C18 alkyl alkoxy ester (AExS), wherein x is from 1-30. Other suitable anionic surfactants can be found in International Publication WO98/39403, Surface Active Agents and Detergents (Vol. 1, & II, by Schwartz, Perry and Berch), and U.S. Pat. Nos. 3,929,678, 6,020,303, 6,060,443, 6,008,181, International Publications WO 99/05243, WO 99/05242 and WO 99/05244, the relevant disclosures of which are incorporated herein by reference.
In another embodiment, the co-surfactant can comprise a cationic surfactant. Suitable cationic surfactants include, for example, those having long-chain hydrocarbyl groups. Examples include ammonium surfactants such as alkyltrimethylammonium halogenides, and surfactants having the formula [R2(OR3)y][R4(OR3)y]2R5N+X−, wherein R2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R3 is independently selected from the group consisting of —CH2CH2—, CH2CH(CH3)—, CH2(CH(CH2OH))—, CH2CH2CH2—, and mixtures thereof; each R4 is selected from the group consisting of C1-C4 alkyl, C1-C4 hydroxyalkyl, benzyl ring structures formed by joining the two R4 groups, —CH2CHOH—CHOHCOR6CHOHCH2OH wherein R6 is any hexose or hexose polymer having a molecular weight less than about 1,000, and hydrogen, when y is not 0; R5 is the same as R4 or is an alkyl chain wherein the total number of carbon atoms of R2 plus R5 is not more than about 18; each y is from 0 to about 10 and the sum of the y values is from 0 to about 15; and X is any compatible anion.
Certain quaternary ammonium surfactant may also be suitable as cationic co-surfactants, and examples of those are described in International Publication WO 98/39403. Examples of suitable quaternary ammonium compounds include coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl di methyl hydroxyethyl ammonium chloride or bromide; C12-15 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl di methyl (ethenoxy) 4 ammonium chloride or bromide. Other cationic surfactants have been described in U.S. Pat. Nos. 4,228,044, 4,228,042, 4,239,660 4,260,529 6,136,769, 6,004,922, 6,022,844, and 6,221,825, International Publications WO 98/35002, WO 98/35003, WO 98/35004, WO 98/35005, WO 98/35006, and WO 00/47708, as well as European Patent Application EP 000,224. When included herein, the surfactant/detergent and the cleaning/treatment compositions of the present invention can comprise, for example, from about 0.2 wt. % to about 25 wt. %, preferably from about 1 wt. % to about 8 wt. % by weight of cationic surfactants.
In certain embodiments, co-surfactants can comprise nonionic surfactants. Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable, with the polyethylene oxide condensates being preferred. They include the condensation products of alkyl phenols having an alkyl group having about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight-chain or branched-chain configuration, with alkylene oxide. In particular embodiments, the ethylene oxide is present in an amount of from about 2 to about 25 moles (e.g., from about 3 to about 15 moles) of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type include IgepalTM C0-630 (The GAF Corp.), TritonTM X-45, X-114, X-100 and X-102 (Dow Chemicals). These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates).
Moreover, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide are suitable nonionic co-surfactants. The alkyl chain of the aliphatic alcohol can be straight or branched, primary or secondary, and generally can contain about 8 to about 22 (e.g., about 8 to about 20, or about 10 to about 18) carbon atoms with about 2 to about 10 moles (e.g., about 2 to about 5 moles) of ethylene oxide per mole of alcohol present in the condensation products. Examples of commercially available nonionic surfactants of this type include TergitolTM 15-S-9, TergitolTM 24-L-6 NMW (Union Carbide); NeodolTM 45-9, NeodolTM 23-3, NeodolTM 45-7, NeodolTM 45-5 (Shell Chemical), KyroTM EOB (Procter & Gamble), and Genapol LA 030 or 050 (Hoechst).
Further examples of nonionic co-surfactants include C12-C18 alkyl ethoxylates (e.g., NEODOL® nonionic surfactants (Shell)), C6-C12 alkyl phenol alkoxylates wherein the alkoxylate units are a mixture of ethyleneoxy and propyleneoxy units, C12-C18 alcohol and C6-C12 alkyl phenol condensates with ethylene oxide/propylene oxide block alkyl polyamine ethoxylates (e.g., PLURONIC® (BASF)), C14-C22 mid-chain branched alcohols as described in U.S. Pat. No. 6,150,322, C14-C22 mid-chain branched alkyl alkoxylates, BAEx, wherein x is from 1-30, as described in U.S. Pat. Nos. 6,153,577, 6,020,303 and 6,093,856, alkylpolysaccharides as described in U.S. Pat. No. 4,565,647, alkylpolyglycosides as described in U.S. Pat. No. 4,483,780 and U.S. Pat. No. 4,483,779, polyhydroxy detergent acid amides as described in U.S. Pat. No. 5,332,528, or ether capped poly(oxyalkylated) alcohol surfactants as described in U.S. Pat. No. 6,482,994 and International Publication WO 01/42408.
Semi-polar nonionic surfactants can also be suitable. They include, e.g., water-soluble amine oxides containing 1 alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from alkyl or hydroxyalkyl moieties containing about 1 to about 3 carbon atoms, water-soluble phosphine oxides containing 1 alkyl moiety of about 10 to about 18 carbon atoms and 2 moieties selected from alkyl or hydroxyalkyl moieties containing about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing 1 alkyl moiety of about 10 to about 18 carbon atoms and a moiety selected from alkyl or hydroxyalkyl moieties of about 1 to about 3 carbon atoms. Semi-polar nonionic surfactants have been described in, e.g., International Publication WO 01/32816, U.S. Pat. Nos. 4,681,704 and 4,133,779.
Moreover, alkylpolysaccharides, such as those described in U.S. Pat. No. 4,565,647, having a hydrophobic group containing about 6 to about 30 carbon atoms (e.g., from about 10 to about 16 carbon atoms) and a polysaccharide, can also be suitable semi-polar nonionic co-surfactants. Others have been described in, for example, International Publication WO 98/39403. When included herein, the cleaning/treatment compositions of the present invention can comprise, for example, about 0.2 wt. % or more (e.g., about 1 wt. % or more, about 5 wt. % or more, or about 8 wt. % or more) of such semi-polar nonionic surfactants. For example, the cleaning compositions of the invention can comprise about 0.2 wt. % to about 15 wt. % (e.g., about 1 wt. % to about 10 wt. %) of semi-polar nonionic surfactants.
In certain embodiments, the co-surfactants comprises ampholytic surfactants. Ampholytic surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight- or branched-chain. One of the aliphatic substituents can contain at least about 8 carbon atoms (e.g., from about 8 to about 18 carbon atoms), and at least another contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, or sulfate. Ampholytica surfactants have been described in, for example, U.S. Pat. No. 3,929,678. When included therein, a cleaning composition of the invention can comprise, for example, about 0.2 wt. % to about 15 wt. % (e.g., about 1 wt. % to about 10 wt. %) of ampholytic surfactants.
In certain other embodiments, especially in personal care cleaning/treatment compositions, zwitterionic surfactants are included as co-surfactants. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. Zwitterionic surfactants have been described in, for example, U.S. Pat. No. 3,929,678. When included therein, a surfactant/detergent or cleaning/treatment composition of the invention can comprise, for example, about 0.2 wt. % to about 15 wt. % (e.g., about 1 wt. % to about 10 wt. %) of zwitterionic surfactants.
In further embodiments, primary or tertiary amines can be included as co-surfactants. Suitable primary amines include amines according to the formula R1NH2 wherein R1 is a C6-C12, preferably C6-C10, alkyl chain; or R4X(CH2)n, wherein X is —O—, —C(O)NH— or —NH—, R4 is a C6-C12 alkyl chain, n is between 1 to 5 (e.g., 3). The alkyl chain of R1 can be straight or branched, and can be interrupted with up to 12, but preferably less than 5 ethylene oxide moieties. Preferred amines include n-alkyl amines, selected from, for example, 1-hexylamine, 1-octylamine, 1-decylamine and laurylamine, C8-C10 oxypropylamine, octyloxypropylamine, 2-ethylhexyl-oxypropylamine, lauryl amido propylamine or amido propylamine. Suitable tertiary amines include those having the formula R1R2R3N wherein R1 and R2 are C1-C8 alkylchains, R3 is either a C6-C12, preferably C6-C10, alkyl chain, or R3 is R4X(CH2)n, whereby X is —O—, —C(O)NH— or —NH—, R4 is a C4-C12, n is between 1 and 5 (e.g., 2, 3, or 4), R5 is H or C1-C2 alkyl, and x is between 1 and 6. R3 and R4 may be linear or branched. The alkyl chain of R3 can be interrupted with up to 12, but preferably less than 5, ethylene oxide moieties. Preferred tertiary amines include, for example, 1-hexylamine, 1-octylamine, 1-decylamine, 1-dodecylamine, n-dodecyldimethylamine, bishydroxyethylcoconutalkylamine, oleylamine(7)ethoxylated, lauryl amido propylamine, and cocoamido propylamine. Other useful detersive surfactants have been described in the prior art, for example, in U.S. Pat. Nos. 3,664,961, 3,919,678, 4,222,905, and 4,239,659.
In some embodiments, the detergent/cleaning composition of the invention comprises greater than about 5 wt. % anionic surfactant and/or less than about 25 wt. % nonionic surfactant. For example, the composition comprises greater than about 10 wt. % anionic surfactants. In another example, the composition comprises less than 15%, more preferably, less than 12% nonionic surfactants.
The total amount of surfactants included in a cleaning composition of the invention is typically about 0.1 wt. % or more (e.g., about 1 wt. % or more, about 10 wt. % or more, about 25 wt. % or more, about 50 wt. % or more, about 60 wt. % or more, about 70 wt. % or more). An exemplary cleaning composition of the invention comprises about 0.1 wt. % to about 80 wt. % total surfactants (e.g., about 1 wt. % to about 50 wt. %, about 10 wt. % to about 40 wt. %, about 20 wt. % to about 35 wt. %) of total surfactants, including the microbially produced branched fatty alcohols and/or derivatives thereof and co-surfactants.
One criteria based on which to the type(s) and amount(s) of surfactants to be included in cleaning compositions can be determined is compatibility with the enzyme components present in the cleaning compositions. For example, in liquid or gel compositions, the cleaning composition (including all the surfactants, which are, for example, pre-formulated into a surfactant package) is prepared such that it promotes, or at least does not degrade, the stability of any enzyme in the cleaning composition.
A surfactant composition of the present invention, or a surfactant package, which can be formulated and subsequently included in a cleaning composition, can be in any form, for example, a liquid; a solid such as a powder, granules, agglomerate, paste, tablet, pouches, bar; a gel; an emulsion; or in a suitable form to be delivered in dual-compartment containers. The composition can also be formulated into a spray or foam detergent, premoistened wipes (e.g., the cleaning composition in combination with a nonwoven material as described, for example, in U.S. Pat. No. 6,121,165), dry wipes (e.g., a cleaning composition in combination with a nonwoven material, activated with water by a consumer, as described, for example, in U.S. Pat. No. 5,980,931), and other homogeneous or multiphase consumer cleaning product forms.
Cleaning Compositions
Surfactant compositions comprising branched fatty alcohols and/or derivatives thereof, e.g., sulfate, alkoxylated or alkoxylated sulfate derivatives, are particularly suitable as soil detachment-promoting ingredients of laundry detergents, dishwashing liquids and powders, and various other cleaning compositions. They exhibit good dissolving power especially when faced with greasy soils, and it is particular advantageous that they display the outstanding soil-detaching power even at low washing temperatures.
The branched fatty alcohol/derivative compositions of the present invention can be included or blended into a surfactant package as described above, which comprises about 0.0001 wt. % to about 100 wt. % of one or more branched fatty alcohols and/or derivatives produced by a genetically engineered host cell or microbe. That surfactant package can then be blended into a cleaning composition to impart detergency and cleaning power to the cleaning composition. In alternative embodiments, the branched fatty alcohols and/or derivatives thereof produced by the host cell or microbe can be blended into a cleaning composition directly, in an amount of about 0.001 wt. % or more (e.g., about 0.001 wt. % or more, about 0.1 wt. % or more, about 1 wt. % or more, about 10 wt. % or more, about 20 wt. % or more, or about 40 wt. % or more) based on the total weight of the cleaning composition. For example, the branched fatty alcohols and/or derivatives thereof can be blended into a composition in an amount of about 0.001 wt. % to about 50 wt. % (e.g., about 0.01 wt. % to about 45 wt. %, about 0.1 wt. % to about 40 wt. %, about 1 wt. % to about 35 wt. %). Accordingly, a cleaning composition of the present invention, in either a solid form (e.g., a tablet, granule, powder, or compact), or a liquid form (e.g., a fluid, gel, paste, emulsion, or concentrate) can comprise about 0.001 wt. % to about 50 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof. For example, a cleaning composition of the invention can comprise about 0.5 wt. % to about 44 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof. Preferably, the cleaning composition comprises about 1 wt. % to about 30 wt. % of microbially produced branched fatty alcohols and/or derivatives.
Alternatively, a cleaning composition of the present invention can comprise about 0.001 wt. % to about 80 wt. % of a surfactant package formulated to comprise about 0.001 wt. % to about 100 wt. % of microbially produced branched fatty alcohols and/or derivatives. For example, a cleaning composition of the present invention can comprise about 0.1 wt. % to about 50 wt. % of such a surfactant package. The surfactant package can comprise other surfactants (i.e., co-surfactants), which can include surfactants derived from similar (e.g., microbially-produced surfactant) or different sources (e.g., petroleum-derived surfactants). In a particular embodiment, however, the surfactant package can comprise mostly or exclusively of branched fatty alcohols and/or derivatives produced by a host cell or a microbe as described herein.
Industrial Cleaning Compositions, Household Cleaning Compositions & Personal Care Cleaning Compositions
In certain embodiments, the cleaning composition of the present invention is a liquid or solid laundry detergent composition. In some embodiments, the cleaning composition is a hard surface cleaning composition, wherein the hard surface cleaning composition preferably impregnates a nonwoven substrate. As used herein, “impregnate” means that the hard surface cleaning composition is placed in contact with a nonwoven substrate such that at least a portion of the nonwoven substrate is penetrated by the hard surface cleaning composition. For example, the hard surface cleaning composition preferably saturates the nonwoven substrate. In other embodiments, the cleaning composition of the present invention is a car care composition, which is useful for cleaning various surfaces such as hard wood, tile, ceramic, plastic, leather, metal, and/or glass. In some embodiments, the cleaning composition is a dish-washing composition, such as, for example, a liquid hand dishwashing composition, a solid automatic dishwashing composition, a liquid automatic dishwashing composition, and a tab/unit dose form automatic dishwashing composition.
In further embodiments, the cleaning composition can be used in industrial environments for cleaning of various equipment, machinery, and for use in oil drilling operations. For example, the cleaning composition of the present invention can be particularly suited in environments wherein it comes into contact with free hardness and in compositions that require hardness tolerant surfactant systems, such as when used to aid oil drilling.
In some embodiments, the cleaning composition of the invention can be formulated into personal or pet care compositions such as shampoos, body washes, or liquid or solid soaps.
Common cleaning adjuncts applicable to most cleaning compositions, including, household cleaning compositions, and personal care compositions and the like, include builders, enzymes, polymers, suds boosters, suds suppressors (antifoam), dyes, fillers, germicides, hydrotropes, anti-oxidants, perfumes, pro-perfumes, enzyme stabilizing agents, pigments, and the like. In some embodiments, the cleaning composition is a liquid cleaning composition, wherein the composition comprises one or more selected from solvents, chelating agents, dispersants, and water. In other embodiments, the cleaning composition is a solid, wherein the composition further comprises, for example, an inorganic filler salt. Inorganic filler salts are conventional ingredients of solid cleaning compositions, present in substantial amounts, varying from, for example, about 10 wt. % to about 35 wt. %. Suitable filler salts include, for example, alkali and alkaline-earth metal salts of sulfates and chlorides. An exemplary filler salt is sodium sulfate.
Household cleaning compositions, including, e.g., laundry detergents and household surface cleaners typically comprise certain additional, in some embodiments, more specialized, ingredients or cleaning adjuncts selected from one or more of: bleaches, bleach activators, catalytic materials, suds boosters, suds suppressors (antifoams), diverse active ingredients or specialized materials such as dispersant polymers (e.g., dispersant polymers sold by BASF or Dow Chemicals), silvercare, anti-tarnish and/or anti-corrosion agents, dyes, germicides, alkalinity sources, hydrotropes, anti-oxidants, enzyme stabilizing agents, pro-perfumes, perfumes, solubilizing agents, carriers, processing aids, pigments, and, for liquid formulations, solvents, chelating agents, dye transfer inhibiting agents, dispersants, brighteners, dyes, structure elasticizing agents, fabric softeners, anti-abrasion agents, hydrotropes, processing aids, and other fabric care agents. The cleaning adjuncts particularly useful for household cleaning compositions and the levels of use have been described in, e.g., U.S. Pat. Nos. 5,576,282, 6,306,812 and 6,326,348. A comprehensive list of suitable laundry or other household cleaning adjuncts is, e.g., in International Publication WO 99/05245.
Personal/pet or beauty care cleaning compositions including, e.g., shampoos, facial cleansers, hand sanitizers, bodywash, and the like, can also comprise, in some embodiments, other more specialized adjuncts, including, for example, conditioning agents such as vitamines, silicone, silicone emulsion stabilizing components, cationic cellulose or polymers such as Guar polymers, anti-dandruff agents, antibacterial agents, dispersed gel network phase, suspending agents, viscosity modifiers, dyes, non-volatile solvents or diluents (water soluble or insoluble), foam boosters, pediculocides, pH adjusting agents, perfumes, preservatives, chelants, proteins, skin active agents, sunscreens, UV absorbers, and minerals, herbal/fruit/food extracts, sphingolipids derivatives or synthetic derivatives and clay.
Common Adjuncts
(1) Enzymes
Various known detersive enzymes can be blended into a cleaning composition of the present invention. Suitable enzymes include, e.g., proteases, amylases, lipases, cellulases, pectinases, mannases, arabinases, galactanases, xylanases, oxidases (e.g., laccases), peroxidases, and/or mixtures thereof. They can provide enhanced cleaning performance and/or fabric care benefits. In general, just as the selection of the type and amount of surfactants to be formulated into a cleaning composition should take account of the enzymes therein, the types of enzyme chosen to be included in the composition should take account of the other components in the composition (including the surfactants). Considerations may include, e.g., the pH-optimum of the overall composition, the presence of absence of enzyme stabilization agents, etc. The enzymes should be present in the cleaning compositions in effective amounts.
Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or engineered mutants (e.g., those described in International Publications WO 92/19729, 98/20115, 98/20116, and 98/34946) can also be included. Suitable proteases can be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (as described in International Publications WO 89/06279 and WO 05/103244). Other suitable serine proteases include those from Micrococcineae sp. and those from Cellulonas sp. and variants thereof as, e.g., described in International Publication WO05/052146. Examples of trypsin-like proteases include trypsin (e.g. of porcine or bovine origin) and Fusarium proteases such as those described in International Publications WO 89/06270 and WO 94/25583. Many proteases are commercially available, including, e.g., AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, EsperaseTM, CoronaseTM, PolarzymeTM, KannaseTM (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM, ProperaseTM, PurafectTM, Purafect PrimeTM, Purafect OxPO, FNA, FN2, FN3, and FN4 (Genencor Int'l Inc.).
Suitable lipases include those of bacterial or fungal origin. For example, suitable lipases can be derived from yeast, from genera such as Candida, Kluyvermyces, pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, or derived from a filamentous fungus, such as Acremonium, Aspergillus, Aureobasidum, Cryptococcus, Filobasidium, Fusarium, Humicolar, Magnaporthe, Mucor, Myceliophthora, Neocallimasix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, thermoascus, Thielavia, Tolypocladium, Thermomyces or Trichoderma. Many chemically modified lipases are also suitable, including, for example, those from Humicola, (e.g., a modified lipase from H. lanuginosa as described in EP 258 068 and 305 216, a modified lipase from H. insolens as described in International Publication WO 96/13580), those from Pseudomonas (e.g., a modified lipase from P. alcaligenes, or from P. pseudoalcaligenes as described in EP 218 272, a modified lipase from P. cepacia as described in EP 331 376, a modified lipase from P. stutzeri as described in GB 1,372,034, a modified lipase from P. fluoresces or Pseudomonas sp. strain SD 705, as described in International Publications WO 95/06720 and WO96/27002, a modified lipase from P. wisconsinensis as described in International Publication WO 96/12012), those from Bacillus (e.g. a modified lipase from B. subtilis as described in Dartois et al. Biochemica et Biophysica Acta, 1131, 253-360 (1993)), a modified lipase from B. stearothermophilus as described in JP Application 64/744992, a modified lipase from B. pumilus as described in International Publication WO 91/16422. Other examples are lipase variants include those described in International Publications WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, and EP 407 225 and 260 105.
A number of lipase enzymes, which can be included in a cleaning composition of the invention, are commercially available. They include LipolaseTM, LipolaseTM Ultra and LipexTM (Novozymes A/S). Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or engineered mutant amylases can also be suitably included in a cleaning composition of the invention. Amylases include, for example, α-amylases obtained from Bacillus (for example, from a special strain of B. licheniformis as described GB Patent 1,296,839). Various mutant amylases, which can be suitably included in a cleaning composition, have been described in International Publications WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424. A number of amylases, which can be included in a cleaning composition of the present invention, are commercially available. They include DuramylTM, TermamylTM, StainzymeTM, Stainzyme UltraTM, Stainzyme PlusTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (Genencor Int'l Inc.).
Suitable cellulases include those of bacterial or fungal origin. Chemically modified or engineered mutant cellulases can also be suitably included in a cleaning composition of the invention. Cellulases include, for example, those obtained from the genera Bacillus, Pseudomonas, Humicola (e.g., from Humicola insolens), Fusarium (e.g., from Fusarium oxysporum), Thielavia, Acremonium, Myceliophthora, as described in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and International Publication WO 89/09259. Especially suitable cellulases are the alkaline or neutral cellulases that impart color care benefits. Examples of such cellulases include those described in EP 0 495 257, 0 531 372, and International Publications WO 96/11262, WO 96/29397, and WO 98/08940. A number of cellulases, especially those that provide added color care benefits, are commercially available, which can be included in a cleaning composition of the invention, especially in, for example, a laundry detergent composition. Commercially available cellulases include, e.g., RenozymeTM, CellucleanTM, EndolaseTM, CelluzymeTM, and CarezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation).
Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or engineered mutant peroxidases/oxidases can also be suitably included in a cleaning composition of the invention. Useful peroxidases include, for example, those obtained from the genera Coprinus (e.g., a periosidase from C. cinereus and variants thereof as described in International Publications WO 93/24618, WO 95/10602, and WO 98/15257). Commercially available peroxidases include, for example, GuardzymeTM (Novozymes A/S).
Suitable enzymes described above can be present in a cleaning composition of the present invention at levels of about 0.00001 wt. % or higher (e.g., about 0.01 wt % or higher, about 0.1 wt. % or higher, about 0.5 wt. % or higher, or about 1 wt. % or higher). For example, one or more such enzymes can be present in a cleaning composition of the invention in an amount of about 0.00001 wt. % to about 2 wt. % (e.g., about 0.0001 wt. % to about 1 wt. %, about 0.001 wt. % to about 0.5 wt. %) based on the total weight of the cleaning composition. In certain embodiments, the enzyme(s) can be present or used at very low levels, for example, at about 0.001 wt. % or lower. In alternative embodiments, enzyme(s) can be formulated, for example, into a heavier duty laundry detergent composition, at about 0.1 wt. % and higher, for example, at about 0.5 wt. % or higher.
2) Enzyme Stabilizers
In certain embodiments, the cleaning composition of the present invention, which comprises one or more enzymes, further comprises one or more enzyme stabilizers. For example, the enzymes employed in the cleaning composition can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes. Known stabilizing agents include, for example, a polyol such as propylene glycol or a glycerol, a sugar or a sugar alcohol, a lactic acid, a boric acid, a boric acid derivative such as an aromatic borate ester, a phenyl boronic acid derivative such as a 4-formylphenyl boronic acid. The enzyme stabilizers can be incorporated into the cleaning composition according to known methods, such as, for example, those described in International Publications WO 92/19709 and WO 92/19708.
3) Builders
Cleaning compositions of the present invention optionally comprise one or more detergent builders or builder systems. When a builder is used, the subject composition can comprise, e.g., at least about 1 wt. % (e.g., at least about 1 wt. %, at least about 5 wt. %, at least about 10 wt. %, at least about 20 wt. %, at least about 30 wt. %, at least about 40 wt. %, at least about 50 wt. %, or more) of one or more builders. For example, a solid cleaning composition of the present invention can comprise, e.g., about 1 wt. % to about 60 wt. % (e.g., about 5 wt. % to about 50 wt. %, about 10 wt. % to about 40 wt. %, about 15 wt. % to about 30 wt. %) of one or more builders or a builder system. For example, a liquid cleaning composition of the present invention can comprise about 0 wt. % to about 10 wt. % of one or more detergency builders.
Various known builder materials can be used, including, e.g., aluminosilicate materials, silicates, polycarboxylates, alkyl- or alkenyl-succinic acid, and fatty acids, materials such as ethylenediamine tetraacetate, diethylene triamine pentamethyleneacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene tramine pentamethylenephosphonic acid. Particularly, builder materials such as calcium sequestrant materials, precipitating materials, calcium ion-exchange materials, polycarboxylate materials, citrate builder, succinic acid builders, aminocarboxylates, and mixtures thereof are preferred.
Examples of calcium sequestrant builder materials include alkali metal polyphosphates, such as sodium tripolyphosphate and organic sequestrants, and ethylene diamine tetra-acetic acid. Examples of precipitating builder materials include sodium orthophosphate and sodium carbonate. Examples of calcium ion-exchange builder materials include the water-insoluble crystalline or amorphous aluminosilicates, of which zeolites are the best known, e.g., zeolite A, zeolite B (also known as zeolite P), zeolite C, zeolite X, zeolite Y, and also the zeolite P-type as described in, e.g., EP 0 384 070.
Of particular importance are citrate builders, including, for example, citric acid and soluble salts thereof (particularly sodium salt), are polycarboxylate builders of particular importance for heavy duty liquid detergent formulations due to their availability from renewable resources and their biodegradability. Oxydisuccinates are also especially useful in such compositions and combinations. Useful succinic acid builders can also be C5-C20 alkyl and alkenyl succinic acids and salts thereof, including laurylsuccinate, myristylsuccinate, palmitylsuccinate, 2-dodecenylsuccinate, 2-pentadecenylsuccinate. with dodecenylsuccinic acid being particularly preferred.
Suitable polycarboxylate builders include, for example, cyclic compounds, particularly alicyclic compounds, such as those described in U.S. Pat. Nos. 3,308,067, 3,723,322, 3,835,163; 3,923,679; 4,102,903, 4,120,874, 4,144,226, and 4,158,635.
Ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyl oxysuccinic acid, various alkali metal, ammonium, and substituted ammonium salts of poly acetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, and polycarboxylates such as mellitic acid, succinic acid, oxy-disuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxy-succinic acid, and soluble salts thereof can be used as builders. Other nitrogen-containing, phospho-free aminocarboxylates are sometimes used. Specific examples include ethylene diamine disuccinic acid and salts thereof (ethylene diamine disuccinates, EDDS), ethylene diamine tetraacetic acid and salts thereof (ethylene diamine tetraacetates, EDTA), and diethylene triamine penta acetic acid and salts thereof (diethylene triamine penta acetates, DTPA). In particular embodiments of a liquid composition, 3,3-dicarboxy-4-oxa-1,6-hexanedioates and related compounds as described in U.S. Pat. No. 4,566,984 can be suitable.
4) Chelating Agents
Cleaning compositions of the present invention can optionally comprise one or a mixture of more than one copper, iron and/or manganese chelating agents. When such an agent is used, the subject cleaning composition can comprise, for example, about 0.005 wt. % or more (e.g., about 0.01 wt. % or more, about 1 wt. % or more, about 5 wt. % or more, about 10 wt. % or more) chelating agents. For example, a cleaning composition of the invention comprises about 0.005 wt. % to about 15 wt. % (e.g., about 0.01 wt. % to about 12 wt. %, about 0.1 wt. % to about 10 wt. %, about 1 wt. % to about 8 wt. %, about 2 wt. % to about 6 wt. %) chelating agents.
Suitable chelating agents include, e.g., amino carboxylates, amino phosphonates, polyfunctionally-substituted aromatic chelating agents, or mixtures, which are capable of removing copper, iron, or manganese ions from washing mixtures by forming soluble chelates.
Amino carboxylates include, for example, ethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates, nitrilotriacetates, ethylenediamine tetraproprionates, triethylenetetraamine-hexacetates, diethylenetriamine penta-acetates, and ethanoldiglycines, alkali metal, ammonium, and substituted ammonium salts thereof.
Amino phosphonates are selectively used in cleaning compositions because they increase the amount of total phosphorus. For some applications wherein the amount of total phosphorus in a cleaning composition is limited, amino phosphonates may not be a suitable chelating agent or should be used in low amounts. Amino phosphonates include, e.g., ethylenediamine tetrakis (methylenephosphonates). The amino phosphonates preferably do not contain alkyl or alkenyl groups with more than about 6 carbon atoms.
Suitable polyfunctionally-substituted aromatic chelating agents have been described in, e.g., U.S. Pat. No. 3,812,044. Exemplary polyfunctionally-substituted aromatic chelating agents include a dihydroxydisulfobenzene, such as a 1,2-dihydroxy-3,5-disulfobenzene.
In some embodiments, biodegradable chelators can be included in a cleaning composition of the invention. An exemplary biodegradable chelator is ethylenediamine disuccinate (“EDDS”), especially the [S,S] isomer as described in U.S. Pat. No. 4,704,233.
The compositions herein may also contain water-soluble methyl glycine diacetic acid (MGDA) salts (or acid form) as a chelant or co-builder useful with, for example, insoluble builders such as zeolites, layered silicates and the like.
5) Hydrotropes
Hydrotropes can be optionally included in cleaning compositions of the present invention to improve the physical and chemical stability of the compositions. Suitable hydrotropes include sulfonated hydrotropes, which include, for example, alkyl aryl sulfonates, or alkyl aryl sulfonic acids. Alkyl aryl sulfonates can be sodium, potassium, calcium, or ammonium xylene sulfonates; sodium, potassium, calcium, or ammonium toluene sulfonates; sodium, potassium, calcium, or ammonium euraene sulfonates; sodium, potassium, calcium, or ammonium substituted or unsubstituted naphthalene sulfonates, and mixtures thereof. Preferred among these are the sodium salts. Alkyl aryl sulfonic acids can be xylenesulfonic acid, toluenesulfonic acid, cumenesulfonic acid, substituted or unsubstituted naphthalenesulfonic acid, or salts thereof. In certain embodiments, a mixture of xylenesulfonic acid and p-toluene sulfonate can be used.
If present, a cleaning composition of the present invention comprises hydrotropes in an amount of about 0.01 wt. % or more (e.g., about 0.02 wt. % or more, about 0.05 wt. % or more, about 0.1 wt. % or more, about 1 wt. % or more, about 5 wt. % or more, about 10 wt. % or more, or about 15 wt. % or more). On the other hand, a cleaning composition of the present invention comprises hydrotropes in an amount of no more bout 20 wt. % (e.g., no more than about 20 wt. %, no more than about 15 wt. %, no more than about 10 wt. %, no more than about 5 wt. %, no more than about 1 wt. %). In certain embodiments, the cleaning composition can comprise hydrotropes in an amount of about 0.01 wt. % to about 20 wt. % (e.g., about 0.02 wt. % to about 18 wt. %, about 0.05 wt. % to about 15 wt. %, about 0.1 wt. % to about 10 wt. %, about 1 wt. % to about 5 wt. %), based on the total weight of the cleaning composition.
6) Rheology Modifier
A cleaning composition of the present invention, when in the form of a liquid, can suitably comprise a rheology modifier to provide a matrix that is “shear-thinning”. A shear-thinning fluid, as it is understood by those skilled in the art, is a fluid the viscosity of which decreases as shear is applied. Thus, at rest, for example, during storage or shipping of a composition, the liquid matrix of the composition preferably has a relatively high viscosity. When shear is applied to the composition, however, such as in the act of pouring or squeezing the composition from its container, the viscosity of the matrix is lowered to the extent that dispensing of the fluid product is easily and readily accomplished.
Various materials that are capable of forming shear-thinning fluids when combined with water or other aqueous liquids are known. One type of useful structuring agent for this purpose comprises non-polymeric (except for conventional alkoxylation) crystalline hydroxy-functional materials that can form thread-like structuring systems throughout the liquid matrix when crystallized within the matrix in situ. Such materials include, e.g., crystalline hydroxyl-containing fatty acids, fatty esters, or fatty waxes. Specific examples of crystalline hydroxyl-containing rheology modifiers include castor oil and derivatives. Preferred are hydrogenated castor oil derivatives such as hydrogenated castor oil and hydrogenated castor wax. A number of these materials are commercially available, including, for example, THIXCIN® (Elementis Specialties), 1,4-di-O-benzyl-D-Threitol in the R,R, and S,S forms and any mixtures, optically active or not, and others described in, for example, U.S. Pat. No. 6,080,708 and International Publication WO 02/40627.
Suitable polymeric rheology modifiers include those of the polyacrylate, polysaccharide or polysaccharide derivative type. Polysaccharide derivatives typically used as rheology modifiers comprise polymeric gum materials. Such gums include pectine, alginate, arabinogalactan, carrageenan, gellan gum, xanthan gum and guar gum. Another suitable rheology modifier is a combination of a solvent and a polycarboxylate polymer. The solvent can be, for example, an alkylene glycol, more preferably dipropy glycol. For example, the solvent can comprise a mixture of dipropyleneglycol and 1,2-propanediol, with a ratio of dipropyleneglycol to 1,2-propanediol being about 3:1 to about 1:3 (e.g., about 1:1). The polycarboxylate polymer can be, e.g., a polyacrylate, polymethacrylate, or mixtures thereof. The polyacrylate can be a copolymer of unsaturated mono- or di-carbonic acid and 1-30C alkyl ester of the (meth) acrylic acid, or a polyacrylate of unsaturated mono- or di-carbonic acid and 1-30C alkyl ester of the (meth) acrylic acid. Some of these polymers are commercially available, for example, under the tradename Carbopol® Aqua 30 (Lubrizol, Wickliffe, Ohio).
The rheology modifiers can be present at a level of about 0.5 wt. % to about 15 wt. % (e.g., about 1 wt. % to about 12 wt. %, about 2 wt. % to about 9 wt. %), based on the total weight of the cleaning composition. The polycarboxylate polymer is suitably present at a level of about 0.1 wt. % to about 10 wt. % (e.g., about 1 wt. % to about 8 wt. %, about 1.5% to about 6 wt. %, about 2 wt. % to about 5 wt. %) in the cleaning composition.
6) Solvents or Solvent Systems
A cleaning composition of the invention can be in a liquid form, wherein one or more suitable solvents or solvent systems are included. Suitable solvents include water, lipophilic fluids, or organic solvents. Examples of suitable lipophilic fluids include siloxanes, other types of silicones, hydrocarbons, glycol ethers, glycerine derivatives such as glycerine ethers, perfluorinated amines, perfluorinated and hydrofluoroether solvents, low-volatility nonfluorinated organic solvents, diol solvents, other environmentally-friendly solvents and mixtures. Particularly suitable solvents include low molecular weight primary and secondary alcohols, such as methanol, ethanol, propanil, or isopropanol. Monohydric alcohols, e.g., polyols containing about 2 to about 6 carbon atoms, and/or about 2 to about 6 hydroxy groups (e.g., propylene glycol, ethylene glycol, glycerin, and 1,2-propanediol) are also suitable.
Solvents can be absent, for example, from anhydrous solid embodiments of the cleaning compositions of the invention. But in a liquid cleaning composition, they are typically present at levels of bout 0.1 wt. % to about 98 wt. % (e.g., about 1 wt. % to about 90 wt. %, about 10 wt. % to about 80 wt. %, about 20 wt. % to about 75 wt. %).
7) Organic Sequestering Agent
A cleaning composition of the invention can optionally comprise about 0.01 wt. % to about 1.0 wt. % of an organic sequestering agent. Non-limiting example of organic sequestering agent include nitriloacetic acid, EDTA, organic phosphonates, sodium citrate, sodium tartrate monosuccinate, sodium tartrate disuccinate, and mixture thereof.
Certain adjuncts are particularly suitable for laundry/household cleaning applications as compared to for personal/beauty care cleaning compositions, while other adjuncts are vise versa. Certain adjuncts are categorized and described below as particularly suitable for the former or the latter, but that categorization is not meant to be exclusive in that adjuncts that are suitable for laundry/household cleaning compositions can be included in personal/beauty care cleaning compositions and vise versa as appropriate.
Adjuncts Particularly Suitable for Laundry/Household Applications
1) Bleach System
A bleach system suitable for use herein can contain one or more bleaching agents. Suitable bleaching agents include, e.g., catalytic metal complexes, activated peroxygen sources, bleach activators, bleach boosters, photobleaches, bleaching enzymes, free radical initiators, and hyohalite bleaches. Suitable activated peroxygen sources include, e.g., preformed peracids, a hydrogen peroxide source with a bleach activator, or a mixture thereof. Suitable preformed peracids include, e.g., percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof. Suitable sources of hydrogen peroxide include, e.g., perborate compounds, percarbonate compounds, perphosphate compounds and mixtures. Suitable types and levels of activated peroxygen sources are described in, e.g., U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348.
A household cleaning composition of the invention can optionally comprise photobleach, which can be, for example, a xanthene dye photobleach, a photo-initiator, or mixtures thereof. Suitable photobleaches can also catalytic photobleaches and photo-initiators. In certain embodiments, catalytic photobleaches are selected from the group consisting of water soluble phthalocyanines of the formula:




wherein: PC is the phthalocyanine ring system; Me is Zn; Fe(II); Ca; Mg; Na; K; Al—Z1; Si(IV); P(V); Ti(IV); Ge(IV); Cr(VI); Ga(III); Zr(IV); In(III); Sn(IV) or Hf(VI); Z1 is a halide; sulfate; nitrate; carboxylate; alkanolate; or hydroxyl ion; q is 0; 1 or 2; r is 1 to 4; Q1 is a sulfur or carboxyl group; or a radical of the formula: —SO2X2—R1—X3+; —O—R1—X3+; or —(CH2), —Y1+; in which R1 is a branched or unbranched C1-C8 alkylene; or 1,3- or 1,4-phenylene; X2 is —NH—; or —N—C1-C5 alkyl; X3+ is a group of the formula:





or, in the case where R1═C1-C5 alkylene, also a group of the formula:





Y1+  is a group of the formula:





wherein t is 0 or 1; R2 and R3 independently of one another are C1-C6 alkyl; R4 is C1-C5 alkyl; C5-C7 cycloalkyl or NR7R8; R5 and R6 independently of one another are C1-C5 alkyl; R7 and R8 independently of one another are hydrogen or C1-C5 alkyl; R9 and R10 independently of one another are unsubstituted C1-C6 alkyl or C1-C6 alkyl substituted by hydroxyl, cyano, carboxyl, carb-C1-C6 alkoxy, C1-C6 alkoxy, phenyl, naphthyl or pyridyl; u is from 1 to 6; A1 is a unit which completes an aromatic 5- to 7-membered nitrogen heterocycle, which may where appropriate also contain one or two further nitrogen atoms as ring members, and B1 is a unit which completes a saturated 5- to 7-membered nitrogen heterocycle, which may where appropriate also contain 1 to 2 nitrogen, oxygen and/or sulfur atoms as ring members; Q2 is hydroxyl; C1-C22 alkyl; branched C3-C22 alkyl; C2-C22 alkenyl; branched C3-C22 alkenyl and mixtures thereof; C1-C22 alkoxy; a sulfo or carboxyl radical; a radical of the formula:





a branched alkoxy radical of the formula:





an alkylethyleneoxy unit of the formula: -(T1)d—(CH2)b(OCH2CH2)e—B3; or an ester of the formula: COOR18, wherein B2 is hydrogen; hydroxyl; C1-C30 alkyl; C1-C30 alkoxy; —CO2H; —CH2COOH; —SO3-M1OSO3-M1; —PO32−M1; —OPO32−M1; and mixtures thereof; B3 is hydrogen; hydroxyl; —COOH; —SO3-M1; —OSO3-M1 or C1-C6 alkoxy; M1 is a water-soluble cation; T1 is —O—; or —NH—; X1 and X4 independently of one another are —O—; —NH— or —N—C1-C5alkyl; R11 and R12 independently of one another are hydrogen; a sulfo group and salts thereof; a carboxyl group and salts thereof or a hydroxyl group; at least one of the radicals R11 and R12 being a sulfo or carboxyl group or salts thereof, Y2 is —O—; —S—; —NH— or —N—C1-C5alkyl; R13 and R14 independently of one another are hydrogen; C1-C6 alkyl; hydroxy-C1-C6 alkyl; cyano-C1-C6 alkyl; sulfo-C1-C6 alkyl; carboxy or halogen-C1-C6 alkyl; unsubstituted phenyl or phenyl substituted by halogen, C1-C4 alkyl or C1-C4 alkoxy; sulfo or carboxyl or R13 and R14 together with the nitrogen atom to which they are bonded form a saturated 5- or 6-membered heterocyclic ring which may additionally also contain a nitrogen or oxygen atom as a ring member; R15 and R16 independently of one another are C1-C6 alkyl or aryl-C1-C6 alkyl radicals; R17 is hydrogen; an unsubstituted C1-C6 alkyl or C1-C6 alkyl substituted by halogen, hydroxyl, cyano, phenyl, carboxyl, carb-C1-C6 alkoxy or C1-C6 alkoxy; R18 is C1-C22 alkyl; branched C3-C22 alkyl; C1-C22 alkenyl or branched C3-C22 alkenyl; C3-C22 glycol; C1-C22 alkoxy; branched C3-C22 alkoxy; and mixtures thereof; M is hydrogen; or an alkali metal ion or ammonium ion, Z2−  is a chlorine; bromine; alkylsulfate or arylsulfate ion; a is 0 or 1; b is from 0 to 6; c is from 0 to 100; d is 0; or 1; e is from 0 to 22; v is an integer from 2 to 12; w is 0 or 1; and A′′ is an organic or inorganic anion, and s is equal to r in cases of monovalent anions A− and less than or equal to r in cases of polyvalent anions, it being necessary for As− to compensate the positive charge; where, when r is not equal to 1, the radicals Q1 can be identical or different, and where the phthalocyanine ring system may also comprise further solubilising groups.

Other suitable catalytic photobleaches include xanthene dyes, sulfonated zinc phthalocyanine, sulfonated aluminium phthalocyanine, Eosin Y, Phoxine B, Rose Bengal, C.I. Food Red 14, and mixtures. In some embodiment, a photobleach can be a mixture of sulfonated zinc phthalocyanine and sulfonated aluminium phthalocyanine, wherein the weight ratio of sulfonated zinc phthalocyanine to sulfonated aluminium phthalocyanine is greater than 1, greater than 1 but less than about 100, or from 1 to about 4.
Suitable photo-initiators include, e.g., aromatic 1,4-quinones such as anthraquinones and naphthoquinones; alpha amino ketones, particularly those containing benzoyl moieties; alphahydroxy ketones, particularly alpha-hydroxy acetophenones; phosphorus-containing photoinitiators, including monoacyl, bisacyl and trisacyl phosphine oxide and sulphides; dialkoxy acetophenones; alpha-haloacetophenones; trisacyl phosphine oxides; benzoin and benzoin based photoinitiators; and mixtures thereof. Photo-initiators can, e.g., be 2-ethyl anthraquinone; Vitamin K3; 2-sulphate-anthraquinone; 2-methyl 1-[4-phenyl]-2-morpholinopropan-1-one (Irgacure® 907); (2-benzyl-2-dimethyl amino-1-(4-morpholinophenyl)-butan-1-one (Irgacure® 369); (1-[4-(2-hydroxyethoxy)-phenyl]-2 hydroxy-2-methyl-1-propan-1-one) (Irgacure® 2959); 1-hydroxy-cyclohexyl-phenyl-ketone (Irgacure® 184) (Ciba); oligo[2-hydroxy 2-methyl-1-[4(1-methyl)-phenyl]propanone (Esacure® KIP 150) (Lamberti); 2-4-6-(trimethyl-benzoyl)diphenyl-phosphine oxide, bis(2,4,6-trimethylbenzoyl)-phenyl-phosphine oxide (Irgacure® 819); (2,4,6 trimethyl benzoyl)phenyl phosphinic acid ethyl ester (Lucirin® TPO-L (BASF)); and mixtures thereof.
A number of photobleaches are commercially available, including those described above, from, e.g., Aldrich; Frontier Scientific; Ciba; BASF; Lamberti S.p.A; Dayglo Color Corporation; Organic Dyestuffs Corp.
2) Pearlescent Agents
Pearlescent agents are optional but commonly included ingredients of a number of household cleaners, especially, e.g., in hard surface cleaners. They are typically crystalline or glassy solids, transparent or translucent compounds capable of reflecting and/or refracting light to produce a pearlescent effects. For example, they are crystalline particles insoluble in the composition in which they are incorporated. Preferably the pearlescent agents have the shape of thin plates or spheres (which are generally spherical). As commonly practiced in the art, particle sizes are measured across the largest diameter of spheres. Plate-like particles are defined as those wherein the two dimensions of the particle (length and width) are at least 5 times the third dimension (depth or thickness). Other crystal shapes like cubes or needles typically do not display pearlescent effect and thus are not used as pearlescent agents.
Suitable pearlescent agents have D0.99 (sometimes referred to as D99) volume particle size of less than 50 μm. Preferably the pearlescent agents have D0.99 of less than 40 μm, e.g., less than 30 μm. More preferably the particles have volume particle size of greater than 1 μm. The D0.99 is a measure of particle size relating to particle size distribution and meaning in this instance that 99% of the particles have volume particle size of less than 50 μm. Volume particle size and particle size distribution can be measured using conventional methods and equipment, such as, e.g., a Hydro 2000G (Malvern Instruments). The choice of a particle size needs to balance the ease of distribution vs. the efficacy of the pearlescent agent because the smaller the particles, the easier they are suspended, but the lower the efficacy.
Liquid compositions containing less water and more organic solvents will typically have a refractive index that is higher in comparison to the more aqueous compositions. In these compositions, pearlescent agents with high refractive index are preferably included because otherwise the pearlescent agents do not impart sufficient visual perlescence even when introduced at high levels (e.g., more than about 3 wt. %). In liquid compositions containing less water and more organic solvents, the pearlescent agent is preferably one having a refractive index of more than 1.41 (e.g., more than 1.8, more than 2.0. In some embodiments, the difference in refractive index between the pearlescent agent and the cleaning composition or medium, to which pearlescent agent is added, is at least 0.02, or at least 0.2, or at least 0.6.
A liquid cleaning composition may comprise about 0.01 wt. % or more (e.g., about 0.02 wt. % or more, about 0.05 wt. % or more, about 0.1 wt. % or more, about 0.5 wt. % or more, about 1.0 wt. % or more, about 1.5 wt. % or more) of one or more pearlescent agents. Typically, however, the liquid composition comprises no more than about 2 wt. % (e.g., no more than about 1.5 wt. %, no more than about 1.0 wt. %, no more than about 0.5 wt. %) of one or more pearlescent agents. For example, a liquid cleaning composition herein comprises about 0.01 wt. % to about 2.0 wt. % (e.g., about 0.1 wt. % to about 1.5 wt. %) of pearlescent agents.
Suitable pearlescent agents may be organic or inorganic. Organic pearlescent agents include, e.g., monoester and/or diester of alkylene glycols, propylene glycol, diethylene glycol, dipropylene glycol, methylene glycol or tetraethylene glycol with fatty acids containing 6 to 22, preferably about 12 to about 18 carbon atoms, e.g., caproic acid, caprylic acid, 2-ethyhexanoic acid, capric acid, lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, linolenic acid, arachic acid, gadoleic acid, behenic acid, erucic acid, and mixtures.
Inorganic pearlescent agents include mica, metal oxide coated mica, silica coated mica, bismuth oxychloride coated mica, bismuth oxychloride, myristyl myristate, glass, metal oxide coated glass, guanine, glitter, and mixtures thereof.
Organic pearlescent agent such as ethylene glycol mono stearate and ethylene glycol distearate provide pearlescence, but typically only when the composition is in motion. Hence only when the composition is poured will the composition exhibit pearlescence. Inorganic pearlescent materials are preferred as the provide both dynamic and static pearlescence. By dynamic pearlescence it is meant that the composition exhibits a pearlescent effect when the composition is in motion. By static pearlescence it is meant that the composition exhibits pearlescence when the composition is static.
Inorganic pearlescent agents are available as a powder, or as a slurry of the powder in an appropriate suspending agent. Suitable suspending agents include ethylhexyl hydroxy-stearate, hydrogenated castor oil. The powder or slurry of the powder can be added to the composition without the need for any additional process steps.
Optionally, co-crystallizing agents can be used to enhance the crystallization of the organic pearlescent agents. Suitable co-crystallizing agents include but are not limited to fatty acids and/or fatty alcohols having a linear or branched, optionally hydroxyl substituted, alkyl group containing from about 12 to about 22, preferably from about 16 to about 22, and more preferably from about 18 to 20 carbon atoms, such as palmitic acid, linoleic acid, stearic acid, oleic acid, ricinoleic acid, behenyl acid, cetearyl alcohol, hydroxystearyl alcohol, behenyl alcohol, linolyl alcohol, linolenyl alcohol, and mixtures thereof.
3) Perfumes/Fragrances
The term “perfume” as used herein encompasses individual perfume ingredients as well as perfume accords. The perfume ingredients are often premixed to form a perfume accord prior to adding to a cleaning composition. As used herein, the term “perfume” can also include perfume microencapsulates. Perfume microcapsules comprise perfume raw materials encapsulated within a capsule made with materials selected from urea and formaldehyde; melamine and formaldehyde; phenol and formaldehyde; gelatine; polyurethane; polyamides; cellulose ethers; cellulose esters; polymethacrylate; and mixtures thereof. Encapsulation techniques are known and described in, for example, “Microencapsulation”: methods and industrial applications, Benita & Simon, eds. (Marcel Dekker, Inc., 1996).
The perfume ingredients that can be included in a cleaning composition can include various natural and synthetic chemicals. Exemplary perfume ingredients include aldehydes, ketones, esters, natural extracts, natural essences and the like.
Industrial cleaning compositions often do not comprise perfume ingredients. However, perfume ingredients are commonly found in household and personal care cleaning compositions. When present, the level of perfume or perfume accord is, e.g., present in an amount of about 0.0001 wt. % or more (e.g., about 0.01 wt. % or more, about 0.1 wt. % or more, about 0.5 wt. % or more, about 2 wt. % or more), based on the total weight of the cleaning composition. For example, the level of perfume or perfume accord can be present in an amount of about 0.0001 wt. % to about 10 wt. % (e.g., about 0.01 wt. % to about 5 wt. %, about 0.1 wt. % to about 2 wt. %, preferably about 0.02 wt. % to about 0.8 wt. %, or about 0.003 wt. % to about 0.6 wt. %), by weight of the detergent composition. The level of perfume ingredients in a perfume accord, if one exists, is typically from about 0.0001 wt. % to about 99 wt. % by weight of the perfume accord. Exemplary perfume ingredients and perfume accords are disclosed in, for example, U.S. Pat. Nos. 5,445,747, 5,500,138, 5,531,910, 6,491,840, and 6,903,061.
4) Dyes, Colorants, and Preservatives
The cleaning compositions herein can optionally contain dyes, colorants, and/or preservatives, or contain one or more, or none of these components. The dyes, colorants and/or preservatives can be naturally occurring or slightly processed from natural materials, or they can be synthetic. For example, natural-occurring preservatives include benzyl alcohol, potassium sorbate and bisabalol, sodium benzoate, and 2-phenoxyethanol. Synthetic preservatives can be selected from, for example, mildewstate or bacteriostate, methyl, ethyl, and propyl parabens, bisguamidine components (e.g., DantagardTM and/or GlydantTM (Lonza Group)). Midewstate or bacteriostate compounds include, without limitation, KATHON® GC, a 5-chloro-3-methyl-4-isothiazolin-3-one, KATHON® ICP, a 2-methyl-4-isothiazolin-4-one, and a blend thereof, and KATHON® 886, a 5-chloro-2-methyl-4-isothiazolin-3-one (Dow Chemicals); BRONOPOL, a 2-bromo-2-nitropropane 1,3 diol (Boots, Co. Ltd.); DOWICIDETM A, a 1,2-benzoisothiazolin-3-one (Dow Chemicals); and IRGASAN® DP 200, a 2,4,4′-trichloro-2-hydroxydiphenylether (Ciba-Geigy, AG).
Dyes and colorants include synthetic dyes such as Liquitint® Yellow or Blue or natural plant yes or pigments, such as natural yellow, orange, red, and/or brown pigment, such as carotenoids, including, for example, beta-carotene and lycopene. The composition can additionally contain fluorescent whitening agents or bluing agents. Certain dyes can be light sensitive, including for example Acid Blue 145 (Crompton), Hidacid® blue (Hilton, Davis, Knowles & Triconh); Pigment Green No. 7, FD&C Green No. 7, Acid Blue 1, Acid Blue 80, Acid Violet 48, and Acid Yellow 17 (Sandoz Corp.); D&C Yellow No. 10 (Warner Jenkinson).
If present, dyes or colorants are, e.g., present in an amount of about 0.001 wt. % or more (e.g., about 0.002 wt. % or more, 0.01 wt. % or more, 0.05 wt. % or more, 0.1 wt. % or more; 0.5 wt. % or more). Usually, dyes and colorants are present, if at all, in an amount of no more than about 1 wt. % (e.g., no more than about 0.8 wt. %, no more than about 0.5 wt. %, no more than about 0.2 wt. %, no more than about 0.1 wt. %, no more than about 0.01 wt. %). For example, dyes and colorants can be present in a cleaning composition herein in an amount of about 0.001 wt. % to about 1 wt. % (e.g., about 0.01 wt. % to about 0.4 wt. %).
5) Fabric Care Benefit Agents
A household cleaning composition can be a laundry detergent, wherein a preferred optional ingredient can be a fabric care benefit agent. As used herein, “fabric care benefit agent” refers to any material that can provide fabric care benefits such as fabric softening, color protection, pill/fuzz reduction, anti-abrasion, anti-wrinkle, and the like to garments and fabrics, particularly on cotton and cotton-rich garments and fabrics, when an adequate amount of the material is present on the garment/fabric. Non-limiting examples of fabric care benefit agents include cationic surfactants, silicones, poly olefin waxes, latexes, oily sugar derivatives, cationic polysaccharides, polyurethanes and mixtures. Suitable silicones include, e.g., silicone fluids such as poly(di)alkyl siloxanes, especially polydimethyl siloxanes and cyclic silicones.
Polydimethyl siloxane derivatives include, e.g., organofunctional silicones. One embodiment of functional silicone are the ABn type silicones, as described in U.S. Pat. Nos. 6,903,061, 6,833,344, and International Publication WO02/018528. A number of silicones are commercially available, including, e.g., WaroTM and SilsoftTM 843 (GE Silicones). Functionalized silicones or copolymers with one or more different types of functional groups such as amino, alkoxy, alkyl, phenyl, polyether, acrylate, silicon hydride, mercaptopropyl, carboxylic acid, quaternized nitrogen are also suitable as fabric care benefit agents. A number of these are commercially available including, e.g., SM2125, Silwet 7622 (GE Silicones), DC8822, PP-5495, DC-5562 (Dow Chemicals), KF-888, KF-889 (Shin Etsu Silicones); Ultrasil® SW-12, Ultrasil® DW-18, Ultrasil® DW-AV, Ultrasil® Q-Plus, Ultrasil® Ca-I, Ultrasil® CA-2, Ultrasil® SA-I, Ultrasil® PE-100 (Noveon Inc.), Pecosil® CA-20, Pecosil® SM-40, Pecosil® PAN-150 (Phoenix Chemical). Oily sugar derivatives suitable as fabric care benefit agents were described in International Publication WO 98/16538. Olean® is a commercial brand for certain oily sugar derivatives marketed by The Procter and Gamble Co.
Many dispersible polyolefins can be used to provide fabric care benefits. The polyolefins can be in the form of waxes, emulsions, dispersions, or suspensions. Preferably, the polyolefin is a polyethylene, polypropylene, or a mixture. The polyolefin can be partially modified to contain various functional groups, such as carboxyl, alkylamide, sulfonic acid or amide groups. For example, the polyolefin is partially carboxyl modified or oxidized.
Polymer latex can also be used to provide fabric care benefits in a water based cleaning composition. Non-limiting examples of polymer latexes include those described in, e.g., International Publication WO 02/018451. Additional non-limiting examples include the monomers used in producing polymer latexes, such as 100% or pure butylacrylate, butylacrylate and butadiene mixtures with at least 20 wt. % of butylacrylate, butylacrylate and less than 20 wt. % of other monomers excluding butadiene, alkylacrylate with an alkyl carbon chain at or greater than C6, alkylacrylate with an alkyl carbon chain at or greater than C6 and less than 50 wt. % of other monomers, or a third monomer added into monomer systems above.
Cationic surfactants are also useful in this invention. Examples of cationic surfactants have been described in, e.g., U.S. Patent Publication US2005/0164905.
Fatty acids can also be used as fabric care benefit agents. When deposited on fabrics, fatty acids or soaps thereof, provide fabric care benefits (e.g., softness, shape retention) to laundry fabrics. Useful fatty acids (or soaps, such as alkali metal soaps) are the higher fatty acids containing from about 8 to about 24 carbon atoms, more preferably from about 12 to about 18 carbon atoms. Soaps can be made by direct saponification of fats and oils or by the neutralization of free fatty acids. Particularly useful are the sodium and potassium salts of the mixtures of fatty acids derived from coconut oil and tallow. Fatty acids can be from natural or synthetic origin, both saturated and unsaturated with linear or branched chains.
Color care agents are another type of fabric care benefit agent that can be suitably included in a cleaning composition. Examples include metallo catalysts for color maintenance, such as those described in International Publication WO 98/39403.
Fabric care benefit agents, when present in a household cleaning composition such as a laundry detergent composition, can suitably be present at a level of up to about 30 wt. % (e.g., up to about 20 wt. %, up to about 15 wt. %, up to about 10 wt. %, up to about 5 wt. %, up to about 2 wt. %), based on the total weight of the cleaning composition. For example, a cleaning composition of the invention comprises about 1 wt. % to about 20 wt. % (e.g., about 2 wt. % to about 15 wt. %, about 5 wt. % to about 10 wt. %) of one or more fabric care benefit agents.
6) Deposition Aid
As used herein, “deposition aid” refers to any cationic polymer or combination of cationic polymers that significantly enhance the deposition of the fabric care benefit agent onto the fabric during laundering. An effective deposition aid typically has a strong binding capability with the water insoluble fabric care benefit agents via physical forces such as van der Waals forces or non-covalent chemical bonds such as hydrogen bonding and/or ionic bonding.
An exemplary deposition aid is a cationic or amphoteric polymer. Amphoteric polymers have a net cationic charge. The cationic charge density of the polymer can range from about 0.05 milliequivalents/g to about 6 milliequivalents/g. The charge density is calculated by dividing the number of net charge per repeating unit by the molecular weight of the repeating unit. Nonlimiting examples of deposition aids include cationic polysaccharides, chitosan and its derivatives, and cationic synthetic polymers. Specific deposition aids include, e.g., cationic hydroxy ethyl cellulose, cationic starch, cationic guar derivatives, and mixtures. Certain deposition aids are commercially available, including, e.g., the JR 30M, JR 400, JR 125, LR 400 and LK 400 polymers (Amerchol Corp.), Celquat® H200, Celquat® L-200, and the Cato® starch (National Starch and Chemical Co.), and Jaguar Cl 3 and Jaguar Excel (Rhodia, Inc.).
7) Fabric Substantive and Hueing Dye
Dyes can be included in a cleaning composition of the invention, e.g., a laundry detergent. Conventionally, dyes include certain types of acid, basic, reactive, disperse, direct, vat, sulphur or solvent dyes. For inclusion in cleaning compositions, direct dyes, acid dyes, and reactive dyes are preferred. Direct dyes are water-soluble dyes taken up directly by fibers from an aqueous solution containing an electrolyte, presumably due to selective adsorption. In the Color Index system, direct dye refers to various planar, highly conjugated molecular structures that contain one or more anionic sulfonate group. Acid dyes are water soluble anionic dyes that are applied from an acidic solution. Reactive dyes are those containing reactive groups capable of forming covalent linkages with certain portions of the molecules of natural or synthetic fibers. Suitable fabric substantive dyes that can be included in a cleaning composition include, e.g., an azo compound, stilbenes, oxazines and phthalocyanines.
Hueing dyes are another type of dyes that may be present in a household cleaning composition of the invention. Such dyes have been found to exhibit good tinting efficiency during a laundry wash cycle without exhibiting excessive undesirable build up during laundering. Typically, a hueing dye is included in the laundry detergent composition in an amount sufficient to provide a tinting effect to fabric washed in a solution containing the detergent. In one embodiment, the detergent composition comprises, e.g., about 0.0001 wt. % to about 0.05 wt. % (e.g., about 0.001 wt. % to about 0.01 wt. %) of a hueing dye.
8) Dye Transfer Inhibitors
A household cleaning composition of the invention, e.g., a laundry detergent composition, can comprise one or more compounds for inhibiting dye transfer from one fabric to another of solubilized and suspended dyes encountered during fabric laundering operations involving colored fabrics. Exemplary dye transfer inhibitors include polymeric dye transfer inhibiting agents, which are capable of complexing or absorbing the fugitive dyes washed out of dyed fabrics before the dyes have an opportunity to become attached to other articles in the wash. Polymeric dye transfer agents are described in, e.g., International Publication WO 98/39403. Modified polyethyleneimine polymers, such as those described in International Publication WO 00/05334, which are water-soluble or dispersible, modified polyamines can also be used. Other exemplary dye transfer inhibiting agents include, e.g., polyvinylpyrridine N-oxide (PVNO), polyvinyl pyrrolidone (PVP), polyvinyl imidazole, N-vinyl-pyrrolidone and N-vinylimidazole copolymers (PVPVI), copolymers thereof, and mixtures. The amount of dye transfer inhibiting agents in the cleaning composition can be, e.g., about 0.01 wt. % to about 10 wt. % (e.g., about 0.02 wt. % to about 5 wt. %, about 0.03 wt. % to about 2 wt. %).
9) Optional Ingredients
Unless specified herein below, an “effective amount” of a particular adjunct or ingredient is preferably present in an amount of about 0.01 wt. % or more (e.g., about 0.1 wt. % or more, about 0.5 wt. % or more, about 1.0 wt. % or more, about 2.0 wt. % or more), based on the total weight of the detergent composition. Optional adjuncts are usually presented in an amount of no more than about 20 wt. % (e.g., no more than about 15 wt. %, no more than about 10 wt. %, no more than about 5 wt. %, or no more than about 1 wt. %).
Examples of other suitable cleaning adjuncts, one or more of which may be included in a cleaning composition, include, e.g., effervescent systems comprising hydrogen peroxide and catalase; optical brighteners or fluorescers; soil release polymers; dispersants; suds suppressors; photoactivators; hydrolysable surfactants; preservatives; anti-oxidants; anti-shrinkage agents; gelling agents (e.g., amidoamines, amidoamine oxides, gellan gums); anti-wrinkle agents; germicides; fungicides; color speckles; antideposition agents such as celluose derivatives, colored beads, spheres or extrudates; sunscreens; fluorinated compounds; clays; luminescent agents or chemiluminescent agents; anti-corrosion and/or appliance protectant agents; alkalinity sources or other pH adjusting agents; solubilizing agents; processing aids; pigments; free radical scavengers, and mixtures. Suitable materials and effective amounts are described in, e.g., U.S. Pat. Nos. 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101. Mixtures of the above components can be made in any proportion.
10) Encapsulated Composition
A cleaning composition, such as a household cleaning composition including a laundry detergent, a dishwashing liquid, or a surface cleaning composition, of the present invention can optionally be encapsulated within a water soluble film. The water-soluble film can be made from polyvinyl alcohol or other suitable variations, carboxy methyl cellulose, cellulose derivatives, starch, modified starch, sugars, PEG, waxes, or combinations thereof.
In certain embodiment the water-soluble film may comprise other adjuncts such as copolymer of vinyl alcohol and a carboxylic acid, the advantages of which have been described in, for example, U.S. Pat. No. 7,022,656. An exemplary benefit of such encapsulation practice is the improvement of the shelf-life of the pouched composition. Another exemplary advantage is that this practice provides improved cold water (e.g., less than 10° C.) solubility to the cleaning composition. The level of the co-polymer in the film material is at least about 60 wt. % (e.g., about 65 wt. %, about 70 wt. %, about 80 wt. %) by weight. The polymer can have any average molecular weight, preferably about 1,000 daltons to 1,000,000 daltons (e.g., about 10,000 daltons to about 300,000 daltons, about 15,000 daltons to 200,000 daltons, about 20,000 daltons to 150,000 daltons). In certain embodiments, the copolymer present in the film is about 60% to about 98% hydrolysed (e.g., about 80% to 95% hydrolysed), to improve the dissolution of the material. In certain embodiments, the copolymer comprises about 0.1 mol % to about 30 mol % (e.g., about 1 mol % to about 6 mol %) of carboxylic acid. In certain embodiments, the water-soluble film comprises additional co-monomers, including, for example, sulfonates and ethoxylates such as 2-acrylamido-2-methyl-1-propane sulphonic acid. In further embodiments, the film can also comprise other ingredients, including, for example, plasticizers, for example, glycerol, ethylene glycol, diethyleneglycol, propane diol, 2-methyl-1,3-propane diol, sorbitol, and mixtures thereof, additional water, disintegrating aids, fillers, anti-foaming agents, emulsifying/dispersing agents, and/or antiblocking agents. It may be useful that the pouch or water-soluble film itself comprises a detergent additive to be delivered to the wash water, for example organic polymeric soil release agents, dispersants, dye transfer inhibitors. Optionally the surface of the film of the pouch may be dusted with fine powder to reduce the coefficient of friction. Sodium aluminosilicate, silica, talc and amylose are examples of suitable fine powders. Certain water-soluble films are commercially available, for example, those marketed under the tradename M8630TM (Mono-Sol).
Adjuncts Particularly Suitable for Personal Care Applications
1) Hair Conditioning Agents
Cleaning compositions of the invention can comprise, in some embodiments such as, for example, in personal or beauty care applications, certain known conditioning agents. An exemplary conditioning agent especially suitable for personal care compositions such as shampoos, is a silicone or a silicone-containing material. Such materials can be selected from, e.g., non-volatile silicones, siloxane gums and resins, aminofunctional silicones, quaternary silicones, and mixtures thereof with each other and with volatile silicones. Examples of these silicone polymers have been disclosed, for example, in U.S. Pat. No. 6,316,541.
Silicone oils are flowable silicone materials having a viscosity as measured at 25° C. of less than about 50,000 centistokes (e.g., less than about 30,000 centistokes). For example, silicone oils typically have a viscosity of about 5 centistokes to about 50,000 centistokes (e.g., about 10 centistokes to about 30,000 centistokes). Suitable silicone oils include polyalkyl siloxanes, polyaryl siloxanes, polyalkylaryl siloxanes, polyether siloxane copolymers, and mixtures. Other insoluble, non-volatile silicone fluids having hair conditioning properties can also be used. Methods of making microemulsions of silicone particles are described in the art, including, e.g., the technique described in U.S. Pat. No. 6,316,541. The silicone may, e.g., be a liquid at ambient temperatures, so as to be of a suitable viscosity to enable the material itself to be readily emulsified to the required particle size of about 0.15 microns or less.
The amount of silicone incorporated into a cleaning composition of the invention may depend on the type of composition and the particular silicone materials used. A preferred amount is about 0.01 wt. % to about 10 wt. %, although these limits are not absolute. The lower limit is determined by the minimum level to achieve acceptable conditioning for a target consumer group and the upper limit by the maximum level to avoid making the hair and/or skin unacceptably greasy. The activity of the microemulsion can be adjusted accordingly to achieve the desired amount of silicone or a lower level of the preformed microemulsion can be added.
The microemulsion of silicone oil may be further stabilized by sodium lauryl sulfate or sodium lauryl ether sulfate with 1-10 moles of ethoxylation. Additional emulsifier, preferably chosen from anionic, cationic, nonionic, amphoteric and zwitterionic surfactants, and mixtures thereof may be present. The amount of emulsifier will typically be in the ratio of about 1:1 to about 1:7 parts by weight of the silicone, although larger amounts of emulsifier can be used, e.g., in about 5:1 parts by weight of the silicone or more. Use of these emulsifiers may be necessary to maintain clarity of the microemulsion if the microemulsion is diluted prior to addition to the personal care cleaning composition. The same detersive surfactants in the cleaning composition can also serve as the emulsifier in the preformed microemulsion.
The silicone microemulsion may be further stabilized using an emulsion polymerization process. A suitable emulsion polymerization process has been described by, for example, U.S. Pat. No. 6,316,541. A typical emulsifier is TEA dodecyl benzene sulfonate which is formed in the process when triethanolamine (TEA) is used to neutralize the dodecyl benzene sulfonic acid used as the emulsion polymerization catalyst. It has been found that selection of the anionic counterion, typically an amine, and/or selection of the alkyl or alkenyl group in the sulfonic acid catalyst can further improve the stability of the microemulsion in the shampoo composition. Examples of preferred amines include, without limitation, triisopropanol amine, diisopropanol amine, and aminomethyl propanol.
2) Pearlescent Agents
Pearlescent agents, such as those described herein above can be suitably included in a personal care cleaning composition such as a shampoo. They are defined, for the purpose of the present disclosure, as materials which impart to a composition the appearance of mother of pearl. Pearlescence is produced by specular reflection of light. Light reflected from pearl platelets or spheres as they lie essentially parallel to each other at different levels in the composition creates a sense of depth and luster. Some light is reflected off the pearlescent agent, and the remainder will pass through the agent, which may pass directly through or be refracted. Reflected, refracted light produces a different colour, brightness and luster.
3) Cationic Cellulose or Guar Polymer
Cleaning compositions of the present invention can further contain a cationic polymer to aid the deposition of the silicone oil component and enhance conditioning performance. Non limiting examples of such polymers are described in the CTFA Cosmetic Ingredient Dictionary, 3rd ed, Estrin, Crosley, & Haynes eds., (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C. (1982)). Suitable cationic polymers include polysaccharide polymers, such as cationic cellulose derivatives, for example, salts of hydroxyethyl cellulose reacted with trimethyl ammonium substituted epoxide, referred to in the industry (CTFA) as Polyquaternium 10, as well as Polymer LR, JR, JP and KG series polymers (Amerchol Corp.). Other suitable cationic cellulose polymers includes the polymeric quaternary ammonium salts of hydroxyethyl cellulose reacted with lauryl dimethyl ammonium-substituted epoxide referred to in the industry (CTFA) as Polyquaternium 24, available under the tradename Polymer LM-200 (Amerchol Corp). Suitable cationic guar polymers include cationic guar gum derivatives, such as guar hydroxypropyltrimonium chloride, and those described in, for example, U.S. Pat. No. 5,756,720. Certain of these polymers are commercially available, including, for example, Jaguar® Excel (Rhodia Corp.).
When used, the cationic polymers herein are either soluble in the cleaning composition or are soluble in a complex coacervate phase in the cleaning composition formed by the cationic polymer and the anionic, amphoteric and/or zwitterionic detersive surfactant component described hereinbefore. Complex coacervates of the cationic polymer can also be formed with other charged materials in the composition.
Concentrations of the cationic polymer in the composition can range from about 0.01 wt. % to about 3 wt. % (e.g., about 0.05 wt. % to about 2 wt. %, about 0.1 wt. % to about 1 wt. %). Suitable cationic polymers have cationic charge densities of at least about 0.4 meq/gm (e.g., at least about 0.6 meq/gm). Suitable cationic polymers have cationic charge densities of no more than about 5 meq/gm, at the pH of intended use of the cleaning composition. In an exemplary personal care cleaning composition, such as, for example, a shampoo, which generally has a pH range of about 3 to about 9 (e.g., about 4 to about 8). As used herein, “cationic charge density” of a polymer refers to the ratio of the number of positive charges on the polymer to the molecular weight of the polymer. The average molecular weight of suitable cationic guars and cellulose polymers is typically at least about 800,000 daltons. For example, suitable cationic polymers, which can be included in a cleaning composition of the present invention, is one of sufficiently high cationic charge density to effectively enhance deposition efficiency of the solid particle components in the cleaning composition. Cationic polymers comprising cationic cellulose polymers and cationic guar derivatives with cationic charge densities of at least about 0.5 meq/gm and preferably less than about 7 meq/gm are suitable.
Preferably, the deposition polymers give good clarity and adequate flocculation on dilution with water during use, especially when suitable electrolytes including, e.g., sodium chloride, sodium benzoate, magnesium chloride, and magnesium sulfate, are added.
4) Perfumes/Fragrances
Just as perfumes or perfume accords are typically included in a household cleaning composition of the invention, perfumes or perfume accords as described herein (e.g., supra) are often included in a personal care cleaning composition, such as a shampoo or a bodywash composition. The perfume ingredients, which optionally can be formulated into a perfume accord prior to blending or formulating the cleaning composition, can be obtained from a wide variety of natural or synthetic sources. They include, without limitation, aldehydes, ketones, esters, and the like. They also include, for example, natural extracts and essences, which can include complex mixtures of ingredients, such as orange oils, lemon oils, rose extracts, lavender, musk, patchouli, balsamic essence, sandalwood oil, pine oil, cedar, and the like. The amount of perfume to be included in a cleaning composition of the invention can vary, for example, from about 0.0001 wt. % to about 2 wt. % (e.g., about 0.01 wt. % to about 1.0 wt. %, about 0.1 wt. % to about 0.5 wt. %), based on the total weight of the cleaning composition.
5) Sensory Indicators—Silica Particles
Optionally, in a personal care cleaning composition of the invention, various sensory indicators can be included. These agents provide a change in sensory feel after an appropriate usage time, allowing for easy and precise recognition for the appropriate time of washing. For example, these agents are particularly suitable for cleaning compositions such as hand cleansers. An exemplary type of sensory indicators are silica particles. The properties of the silica particle may be adjusted to provide the desired end point in time.
Various silica particles are commercially available, including, for example, those made and distributed by INEOS Silicas Ltd. These particles have also been described in, for example, U.S. Pat. No. 6,165,510, U.S. Patent Publication 2003/0044442.
Silica particles can be present in an amount that can initially be felt by hands when starting washing with the cleaning composition. In one embodiment, the amount of silica particles is about 0.05 wt. % to about 8 wt. %. In some embodiments, suitable silica particles can have an initial average diameter of about 50 μm to about 600 μm (e.g., about 180 to about 420 μm). In some embodiments, silica particles can further comprise color or pigment on the surface. In other embodiments, suitable silica particles diminish in size and cannot be felt by users during washing before about 5 min, about 2 min, about 30 sec, about 25 sec, about 20 sec, about 15 sec, about 10 sec, about 5 sec, about 5 to about 30 sec, or about 10 to about 30 sec.
Silica particles can also, in addition to providing sensory indications, improve the dispensing of the cleaning composition. For example, by including these particles, the cleaning composition, such as a liquid hand cleaner or a shampoo, may achieve a desirable thickness such that it is easier to be dispensed with a pump.
It is often desirable to regulate the viscosity of a composition comprising silica particles, however. Addition of glycerin has been found to be an effective approach to achieve this regulation. Glycerin is typically added to a composition comprising silica particles in an amount of at least about 1 wt. % (e.g., about 2 wt. %, about 2.5 wt. %, about 3 wt. %, about 4 wt. %, about 5 wt. %, or about 6 wt. %), based on the total weight of the cleaning composition. In some embodiments, glycerin is added in an amount of less than about 10 wt. % (e.g., less than about 8 wt. %, less than about 6 wt. %, less than about 4 wt. %, less than about 2 wt. %). The addition of glycerin may, in certain embodiments, help prevent clogging of pumps.
6) Suspension Agents—Viscosity Control
Cleaning compositions of the invention can also include a suspending agent that allows the particulate matters therein, e.g., the silica particles, to remain suspended. Suspending agents are materials that are capable of increasing the ability of the composition to suspend material. Examples of suspending agents include, e.g., synthetic structuring agents, polymeric gums, polysaccharides, pectin, alginate, arabinogalactan, carrageen, gellan gum, xanthum gum, guar gum, rhamsan gum, furcellaran gum, and other natural gum. An exemplary synthetic structuring agent is a polyacrylate. An exemplary acrylate aqueous solution used to form a stable suspension of the solid particles is manufactured by Lubrizol as CARBOPOLTM resins, also known as CARBOMERTM, which are hydrophilic high molecular weight, crosslinked acrylic acid polymers. Other polymers suitable as suspension agents include, e.g., CARBOPOLTM Aqua 30, CARBOPOLTM 940 and CARBOPOLTM 934.
The suspending agents can be used alone or in combination. The amount of suspending agent can be any amount that provides for a desired level of suspending ability. In certain embodiment, the suspending agent is present in an amount of about 0.01 wt. % to about 15 wt. % (e.g., about 0.1 wt. % to about 12 wt. %, about 1 wt. % to about 10 wt. %, about 2 wt % to about 5 wt. %) by weight of the cleaning composition.
7) Other Suitable Adjuncts
A number of other adjuncts can be suitable for inclusion in a personal care cleaning composition. Those include, for example, thickeners, such as hydroxylethyl cellulose derivatives (e.g., MethocelTM products, Dow Chemicals; Natrosol® products, Aqualon Ashland; CarbopolTM products, Lubrizol).
Stability enhancers can also be included as suitable adjuncts. They are typically nonionic surfactants, including those having an hydrophilic-lipophilic balance range of about 9-18. These surfactants can be straight chained or branched chained, and they typically containing various levels of ethoxylation/propoxylation. The nonionic surfactants useful in the present invention are preferably formed from a fatty alcohol, a fatty acid, or a glyceride with a C3 to C24 carbon chain, preferably a C12 to C18 carbon chain derivatized to yield a Hydrophilic-Lipophilic Balance (HLB) of at least 9. HLB is understood to mean the balance between the size and strength of the hydrophilic group and the size and strength of the lipophilic group of the surfactant. Suitable adjuncts for personal care cleaning compositions can also include various vitamins, including, for example, vitamin B complex; including thiamine, nicotinic acid, biotin, pantothenic acid, choline, riboflavin, vitamin B6, vitamin B12, pyridoxine, inositol, carnitine, vitamins A, C, D, E, K, and their derivatives.
Further suitable adjuncts may include one or more materials such as antimicrobial agents, antifungal agents, antidandruff agents, dyes, foam boosters, pediculocides, pH adjusting agents, preservatives, proteins, skin active agents, sunscreens, UV absorbers, minerals, herbal/fruit/food extracts, sphingolipid derivatives or synthetic derivatives, and clay.
Examples of Preferred Embodiments
Surfactant compositions of the invention can be formulated or used without substantial post-production processing. This is especially the case if the surfactant composition is applied in industrial settings, for example, in oil industry for oil recovery applications. Because typically minimum purity specification is required in such settings, it is potentially possible to use whole-cell broths. Surfactants comprising microbially-produced branched fatty alcohols and derivatives prepared in accordance with the methods herein are relatively more selective, as compared with conventional chemical surfactants. As such, they are required in small quantities, and effective under a broad range of oil and reservoir conditions. They are also more environmentally friendly in protection of coastal areas from additional damage inflicted by synthetic chemicals, because they are readily biodegradable and have lower toxicity than synthetic surfactants. Potentially an about 30% or more increase in total oil recovery from underground sandstone can be achieved using surfactants comprising microbially produced fatty alcohols and derivatives such as those described herein.
Microbially-produced fatty alcohols, including branched fatty alcohols and derivatives thereof such as those described herein, are also more anaerobic, halotolerant and thermo-tolerant as compared to their petroleum-derived counterparts, making surfactants comprising these fatty alcohols particularly useful for in situ enhanced oil recovery. These surfactants are potent reducers of oil viscosity, making it vastly easier to pump heavy oils from underground sandstone as well as through commercial pipelines for long distances. Microbially-produced fatty alcohols and derivatives and surfactants comprising these materials can also be used to desludge crude oil storage tanks. The branched fatty alcohols and derivates described herein also have improved low temperature properties, and are thus particularly suited for application in low temperature environments such as in the deep sea.
Potentially, suitable host cells can be engineered such that the culture broth not only provide suitable surfactants but also provides biodegradation of hydrocarbons, resulting in microbial remediation of hydrocarbon- and crude oil-contaminated soils. Furthermore, the branched fatty alcohols, derivatives thereof, as well as the surfactants comprising these materials can be used to manage and emulsify hydrocarbon-water mixtures. This capacity to effectively emulsify oil/water mixtures can be utilized in oil spill management.
With more extensive post-production processing, surfactants comprising the branched fatty alcohols and derivatives as described herein can be particularly suitable as food additives or in the health care and cosmetic industries. The branching of these molecules confer added oxidative stability and significantly decreased volatility and vapor pressure. They are also useful as ingredients in various household and personal and/or pet care cleaning compositions, with particular advantages at lower washing temperatures.
In certain embodiments, the invention features a surfactant composition comprising about 0.001 wt. % to about 100 wt. % (e.g., about 0.01 wt. % to about 80 wt. %, about 0.1 wt. % to about 70 wt. %, about 1 wt. % to about 60 wt. %, about 5 wt. % to about 50 wt. %) of one or more microbially produced branched fatty alcohols and/or derivatives thereof. An exemplary surfactant composition of the invention comprises about 0.1 wt. % to about 50 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof. The surfactant composition of the present invention can further comprise one or more other co-surfactants, derived from similar origins (e.g., microbially produced) or different origins (e.g., chemically synthesized, derived from petroleum sources).
In another aspect, the invention pertains to a cleaning composition comprising one or more surfactants comprising branched fatty alcohols and derivatives produced in accordance with the methods described herein. The inventive cleaning composition can be formulated as a solid cleaning composition or as a liquid cleaning composition.
In certain embodiment, the invention provides a cleaning composition comprising about 0.1 wt. % to about 50 wt. % (e.g., about 0.1 wt. % to about 50 wt. %, about 0.5 wt. % to about 45 wt. %, about 1 wt. % to about 40 wt. %, about 5 wt. % to about 35 wt. %, about 10 wt. % to about 30 wt. %) of one or more microbially produced branched fatty alcohols and/or derivatives thereof. An exemplary cleaning composition comprises about 1 wt. % to about 40 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof. In another embodiment, the composition comprises about 2 wt. % to about 20 wt. % of microbially produced branched fatty alcohols and/or derivatives thereof.
In one embodiment, the invention features a liquid cleaning composition comprising (a) about 0.1 wt. % to about 50 wt. % (e.g., about 0.1 wt. % to about 50 wt. %, about 0.5 wt. % to about 45 wt. %, about 1 wt. % to about 40 wt. %, about 5 wt. % to about 35 wt. %, about 10 wt. % to about 30 wt. %) of one or more microbially produced branched fatty alcohols and/or derivatives thereof, (b) about 1 wt. % to about 30 wt. % (e.g., about 2 wt. % to about 25 wt. %, about 5 wt. % to about 20 wt. %) of one or more co-surfactant, (c) about 0 wt. % to about 10 wt. % (e.g., about 0 wt. % to about 10 wt. %, about 0 wt. % to about 8 wt. %, about 0 wt. % to about 5 wt. %, about 0 wt. % to about 2 wt. %) of one or more detergency builders, (d) about 0 wt. % to about 2.0 wt. % (e.g., about 0.0001 wt % to about 1.5 wt. %, about 0.001 wt. % to about 1 wt. %, about 0.01 wt. % to about 0.8 wt. %) of one or more enzymes; (e) about 0 wt. % to about 15 wt. % (e.g., about 0 wt. % to about 12 wt. %, about 0 wt. % to about 10 wt. %, about 0 wt. % to about 8 wt. %, about 0 wt. % to about 5 wt. %) of one or more chelating agents; (f) about 0 wt. % to about 20 wt. % (about 0 wt. % to about 15 wt. %, about 0 wt. % to about 10 wt %, about 0 wt. % to about 5 wt. %) of one or more hydrotropes; (g) about 0 to about 15 wt. % (e.g., about 0 wt. % to about 10 wt. %, about 0 wt. % to about 8 wt. %, about 0 wt. % to about 5 wt. %) of one or more rheology modifiers; (h) about 0 wt % to about 1.0 wt. % (e.g., about 0 wt. % to about 0.8 wt. %, about 0 wt. % to about 0.5 wt. %, about 0 wt. % to about 0.2 wt. %) of one or more organic sequestering agents; and (i) about 0.1 wt. % to about 98 wt. % (e.g., about 0.1 wt. % to about 95 wt. %, about 1 wt. % to about 90 wt. %, about 10 wt. % to about 85 wt. %) of a solvent system comprising water or other suitable solvents.
In another embodiment, the invention features a solid detergent composition comprising (a) about 0.1 wt. % to about 50 wt. % of one or more microbially produced fatty alcohols and/or derivatives thereof, (b) about 1 wt. % to about 30 wt. % of one or more co-surfactants, (c) about 1 wt. % to about 60 wt. % of one or more detergency builders, (d) about 0 wt. % to about 2.0 wt. % of one or more enzymes, (e) about 0 wt. % to about 20 wt. % of one or more hydrotropes, (f) about 10 wt. % to about 35 wt. % of one or more filler salts, (f) about 0 wt. % to about 15 wt. % of one or more chelating agents, and (g) about 0.01 wt. % to about 1 wt. % of one or more organic sequestering agents.
When the cleaning composition is a solid (e.g., a particulate, a granule, a tablet), the composition herein can be in any solid form, such as a granular composition or for example a tablet, flake, extrudate, agglomerate, or granule-containing composition. Alternatively, the detergent composition can be a powder. The composition herein can be made by methods such as dry-mixing, agglomerating, compaction, spray drying of various ingredients comprised in the composition herein, or a combination thereof. The composition herein preferably has a bulk density of from about 300 g/L, 350 g/L, or 450 g/L to 1500 g/L, 1000 g/L, or 850 g/L.
In certain embodiments, a liquid cleaning composition of the present invention is formulated such that during use, the wash water will have a pH of between about 6.5 and about 11.0 (e.g., between about 6.5 to about 11, between about 7.0 to about 8.5).
In one embodiment, the invention provides a dishwashing detergent composition. The dishwashing detergent composition can be formulated for use in hand washing of dishes or for use in automatic dishwashers. A skilled person will appreciate that a detergent composition formulated for use in automatic dishwashers should contain suitable antifoaming agents in order to prevent excessive foaming of the detergent composition within the dishwasher. However, foaming may be desirable when hand washing dishes. Antifoaming agents are known. For example, various silicone antifoam compounds can be used, including a variety of relatively high molecular weight polymers containing siloxane units and hydrocarbyl groups of various types. Other suitable antifoam agents include monocarboxylic fatty acids and soluble salts thereof, high molecular weight fatty esters (e.g., fatty acid triglycerides), fatty acid esters of monovalent alcohols, aliphatic C18-C40 ketones (e.g., stearone), N-alkylated amino triazines (e.g., tri- to hexa-alkylmelamine or di- to tetra-alkyldiamine chlortriazines), propylene oxide, bis-stearic acid amide, and monostearyl di-alkali metal (e.g., sodium, potassium, lithium) phosphate and phosphate esters, amine oxides, alkanolamides, betaines, and mixtures thereof.
In addition the dishwashing detergents can optionally comprise one or more enzymes, gelling agents, abrasive materials, fragrances, solubility enhancers, antideposition agents, e.g., cellulose derivatives. Abrasive materials can be, e.g., pumice, sand, feldspar, corn meal, or mixtures. Antideposition agent can be present in an exemplary cleaning composition in an amount of about 0.1 wt. % to about 5 wt. % (e.g., about 0.1 wt. % to about 2 wt. %).
In certain embodiments, the invention provides a laundry detergent composition comprising, in addition to the microbially produced branched fatty alcohols and/or derivatives thereof as described herein, the co-surfactants and the builders, optionally one or more enzymes, gelling agents, fragrances, antideposition agents, brighteners, anticaking agents, pearlescent agents, fabric softeners, bleach systems, dyes or colorants, preservatives, fabric care benefit agents, hueing dyes, soil release polymers, photoactivators, hydrolysable surfactants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, colored beads, fluorinated compounds, etc.
In certain embodiments, the invention further provides a solid surface cleaning composition. In addition to the microbially produced branched fatty alcohols and/or derivatives thereof as described herein, the co-surfactants and the builders, the surface cleaning composition can further comprise one or more of the optional ingredients including, without limitation, one or more enzymes, gelling agents, fragrances, antideposition agents, pearlescent agents, soil release polymers, germicides, abrasive materials, fungicides and mixtures thereof.
In certain embodiments, the invention also provide a personal and/or pet care cleaning composition comprising one or more microbially produced branched fatty alcohols and/or derivatives thereof, builders, and co-surfactants. Optionally, additional components can be included in the personal and/or pet care cleaning composition, including, for example, conditioners, silicones, silica particles, cationic cellulose or guar polymers, silicone microemulsion stabilizers, enzymes, fatty amphiphiles, germicides, fungicides, anti-dandruff agents, pearlescent agents, foam boosters, pediculocides, pH adjusting agents, UV absorbers, sunscreens, skin active agents, vitamins, minerals, herbal/fruit/food extracts, sphingolipids, sensory indicators, suspension agents, and mixtures thereof.
The invention further provides a method for cleaning a substrate, such as fibers, fabrics, hard surfaces, skin, hair, etc., by contacting the substrate with the cleaning composition of the invention and water. Agitation is preferably provided to enhance cleaning. Suitable means for providing agitation include rubbing by hand or with a brush, sponge, cloth, mop, or other cleaning device, automatic laundry machines, automatic dishwashers, and the like.
EXAMPLES
The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.
Although particular methods are described, one of ordinary skill in the art will understand that other, similar methods also can be used. In general, standard laboratory practices were used, unless otherwise stipulated. For example, standard laboratory practices were used for: cloning; manipulation and sequencing of nucleic acids; purification and analysis of proteins; and other molecular biological and biochemical techniques. Such techniques are explained in detail in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor, N.Y. (2000), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences (1989).
Example 1
Constructing E. coli MG1655 ΔfadE_ΔtonA AAR:kan
This example describes the construction of a genetically engineered microorganism in which the expression of a fatty acid degradation enzyme is attenuated.
The fadE gene of E. coli MG1655 was deleted using the lambda red system described by Datsenko et al., Proc. Natl. Acad. Sci. USA 97: 6640-6645 (2000), with the following modifications:
The following two primers were used to create the deletion of fadE:








Del-fadE-F
(SEQ ID NO: 158)







5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACAT


 


ATTGATTCCGGGGATCCGTCGACC;


and


 








Del-fadE-R
(SEQ ID NO: 159)







5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTT


 


TCCTGTAGGCTGGAGCTGCTTC






The Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 by PCR. The PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in SOC medium at 37° C., the cells were plated on Luria agar plates containing 50 μg/ml of Kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fadE gene was confirmed in some of the colonies by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.
The fadE deletion confirmation primers were:









(SEQ ID NO: 160)



fadE-L2   5′-CGGGCAGGTGCTATGACCAGGAC;



and



 



(SEQ ID NO: 161)



fadE-R1   5′-CGCGGCGTTGACCGGCAGCCTGG






After the fadE deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain with the fadE gene deleted and the KmR marker removed was named E. coli MG1655 ΔfadE, or E. coli MG 1655 D1.
Furthermore, the expression of an outer membrane protein receptor for ferrichrome, colicin M, or phages T1, T5, and phi80 are attenuated.
The tonA gene of E. coli MG1655, which encodes a ferrichrome outer membrane transporter (GenBank Accession No. NP—414692), was deleted from strain E. coli MG1655 D1 of Example 1, using the lambda red system according to Datsenko et al., supra, but with the following modifications:
The primers used to create the deletion of tonA were:








Del-tonA-F
(SEQ ID NO: 162)







5′-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGATATACC


 


AATGATTCCGGGGATCCGTCGACC;


and


 








Del-tonA-R
(SEQ ID NO: 163)







5′-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGAAG


 


GTTGCGG TTGTAGGCTGGAGCTGCTTC






The Del-tonA-F and Del-tonA-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 by PCR. The PCR product obtained in this way was used to transform electrocompetent E. coli MG1655 D1 cells of Example 1 containing pKD46, which cells had been previously induced with arabinose for 3-4 h. Following a 3-hour outgrowth in SOC medium at 37° C., cells were plated on Luria agar plates containing 50 μg/ml of kanamycin. Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the tonA gene was confirmed in some of the colonies by PCR amplification using primers flanking the E. coli tonA gene: tonA-verF and tonA-verR:.










tonA-verF
(SEQ ID NO: 164)









5′-CAACAGCAACCTGCTCAGCAA;



and



 










tonA-verR
(SEQ ID NO: 165)









5′-AAGCTGGAGCAGCAAAGCGTT






After the tonA deletion was confirmed, a single colony was used to remove the KmR marker using the pCP20 plasmid as described by Datsenko et al., supra. The resulting MG1655 E. coli strain having fadE and tonA gene deletions was named E. coli MG1655 ΔfadE_ΔtonA, or E. coli MG1655 DV2
The aar gene encoding Synechococcus elongatus PCC_Synpcc7942—1594 enzyme is integrated into the chromosome with the kanamycin marker directly after the aar sequence.
Example 2
Expression of BKD Homologs and FabH in E. coli 
A branched chain alpha-keto acid dehydrogenase complex from Pseudomonas putida and a FabH from Bacillus subtilis were used to generate two E. coli plasmids for expression. First, the Pseudomonas putida BKD operon was PCT-amplified from Pseudomonas putida F1 genomic DNA. The following primers were used:










P.p.BKDFUsion_F:
(SEQ ID NO: 166)









5′-ATAAACCATGGATCCATGAACGAGTACGCCCC-3′



 










P.pBKDFusion_R:
(SEQ ID NO: 167)









5′-CCAAGCTTCGAATTCTCAGATATGCAAGGCGTG-3′






Using these primers, Pseudomonas putida Pput—1450 (GenBank Accession No. A5W0E08), Pput—1451 (GenBank Accession No. A5W0E9), Pput—1452 (GenBank Accession No. A5W0F0), and Pput—1453 (A5W0F1) were amplified. The PCR product was then cloned into vector pGL10.173B (See, FIG. 8), a plasmid with a pBR322 backbone and a pTrc promoter to drive gene expression. The PCR product was cloned into pGL between BamHI and EcoRI restriction sites. Correct insertion of the PCR product was verified by diagnostic restriction digests. The resulting plasmid was named “pKZ4.” (See, FIG. 7)
To clone E. coli PfabH promoter-B. subtilis fabH1 into a pACYC vector, insert of pDG6 (pCFDuet-E. coli PfabH promoter-B. subtilis fabH1) was subcloned into pACYC vector using NcoI and AvrII restriction sites. The resulting plasmid was named pDG6 (pCFDuet+E. coli PfabH+B. subtilis fabH1). (See, FIG. 6B and FIG. 6C)
E. coli strain MG1655 ΔfadE_ΔtonA, AAR:kan was transformed with pKZ4 and pDG6 (pCFDuet+E. coli PfabH+B. subtilis fabH1). The strain was evaluated for production of branched chain materials using shake flask fermentation. Shake flask fermentation was carried out using Che-9 media. Specifically, cultures of E. coli MG1655 ΔfadE_ΔtonA AAR:kan without plasmids or carrying individual plasmids were used as controls. Seed cultures of E. coli MG1655 ΔfadE_ΔtonA AAR:kan, E. coli MG1655 ΔfadE_ΔtonA AAR:kan+pKZ4, E. coli MG1655 ΔfadE_ΔtonA AAR:kan+pDG6, and E. coli MG1655 ΔfadE_ΔtonA AAR:kan+pKZ4+pDG6 were grown in LB broths supplemented with the appropriate antibiotics. After 4 hours of growth, the cultures were diluted 1:25 in Che-92NBT medium+appropriate selection marker and grown overnight. The cultures were then diluted in 4NBT to a final OD600 ̃0.2. After 6 h of growth, IPTG was added to a final concentration of 1 mM. At 24 h post-induction, 1 mL of culture was extracted with 0.5 mL of methyl tert-butyl ether (MTBE) and subjected to GC/MS analysis. The analysis revealed the production of iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0, and anteiso-C17:0 fatty alcohols. (See, FIG. 4A).
Example 3
Quantification and Identification of Branched Fatty Alcohols
Instrumentation:
The instrument is an Agilent 5975B MSD system equipped with a 30 m×0.25 mm (0.10 μm film) DB-5 column. The mass spectrometer was equipped with an electron impact ionization source. Two GC/MS programs were utilized.
GC/MS program #1: The temperature of the column is held isothermal at 90° C. for 5 min, then is raised to 300° C. with a 25° C./min ramp, and finally stays at 300° C. for 1.6 min. The total run time is 15 min. With this program, the inlet temperature is hold at 300° C. The injector is set at splitless mode. 1 μL of sample is injected for every injection. The carrier gas (helium) is released at 1.0 mL/min. The source temperature of the mass spectrometer is held at 230° C.
GC/MS program #2: The temperature of the column is held isothermal at 100° C. for 3 min, then is raised to 320° C. with 20° C./min, and finally stays isothermal at 320° C. for 5 min. The total run time is 19 min. The injector is set at splitless mode. 1 μl, of sample is injected for every injection. The carrier gas (helium) is released at 1.2 mL/min. The ionization source temperature is set at 230° C.
Samples:
Extracts containing fatty alcohols by the engineered E. coli strains were analyzed on GC/MS. In FIG. 4A chromatograms of the extracts from the mutant strains are compared to those from control strains which only produce straight chain fatty alcohols. The branched fatty alcohol produced are listed: iso-C14:0, iso-C15:0, anteiso-C15:0, iso-C16:0, iso-C17:0, and anteiso-C17:0.
In FIG. 4A top panel, a GC/MS chromatogram of extract from strain E. coli MG1655 ΔfadE ΔtonA AAR:kan+pKZ4+pDG6 (a) and of control strain E. coli MG1655 ΔfadE ΔtonA AAR:kan+pBR322+pCFDuet(b). Both chromatograms were obtained with GC/MS program #2. Compared to the control strain, mutant strain produces branched-chain fatty alcohols, and the peaks representing the branched fatty alcohols are boxed.
GC/MS Semi-Quantitative Analysis:
In addition to the qualitative analysis, semi-quantitative analysis was performed to obtain the ratio between the branched chain compounds and the straight chain isomers. Due to the lack of commercially available standards for branched fatty alcohols, accurate quantitation for the branched chain compounds was challenging. However, by using straight chain standard with the same functional group, the relative quantity or yield of branched-chain fatty alcohols in relation to the yield of their straight-chain counterpart (isomers) were estimated semi-quantitatively. Standard curve quantitation method was applied, wherein standard mixtures with different concentrations were analyzed by the same GC/MS program as the samples. After data acquisition, the instrument response (total ion current) was plotted against the concentrations of the standards. Linear calibration curves were obtained. (See, FIG. 5). The concentration of branched alcohols in a given sample was calculated according to Equation 1: y=ax+b, wherein y is the instrument response for a particular compound in a sample. Slope a and intercept b for this calibration curve were determined by the linear regression of all calibration levels of standard fatty alcohols (FIG. 4A lower panel)×(the concentration of the branched fatty alcohol product in the sample). Accordingly, the relative concentration of branched fatty alcohols in the production mixture was calculated.
The table below lists the compounds used as standards to quantify different branched fatty alcohol compounds.














Alcohol in sample
Standard used for quantitation









Iso-Alc C15:0
Alc C15:0



Anteiso-Alc C15:0
Alc C15:0



Alc C15:0
Alc C15:0



Ald C16:0
Alc C15:0



Alc C16:0
Alc C15:0










Once the titers were obtained for all the fatty alcohol compounds, the ratio between the production of branched chain fatty alcohols and the production of straight chain isomers were calculated according to equation 2:




  
    Percentage
    ⁢
    
      
    
    ⁢
    production
  
  =
  
    
      
        Total
        ⁢
        
          
        
        ⁢
        branched
        ⁢
        
          
        
        ⁢
        chain
        ⁢
        
          
        
        ⁢
        products
        ⁢
        
          
        
        ⁢
        in
        ⁢
        
          
        
        ⁢
        mg
        ⁢
        
          /
        
        ⁢
        L
      
      
        Total
        ⁢
        
          
        
        ⁢
        straight
        ⁢
        
          
        
        ⁢
        chain
        ⁢
        
          
        
        ⁢
        products
        ⁢
        
          
        
        ⁢
        in
        ⁢
        
          
        
        ⁢
        mg
        ⁢
        
          /
        
        ⁢
        L
      
    
    ×
    100
    ⁢
    %
  




Using this method, we were able to semi-quantitatively estimate the amount of branched fatty alcohol yield relative to the straight-chain fatty alcohol yield to be about 48%.
Example 4
Production of Branched Acyl-CoA Precursors
An E. coli strain, MG1655(DE3) ΔfadE::FRT ΔfabH::cat/pDG6 was created, which was tested for its ability to utilize branched-chain substrate molecules to create branched-chain fatty precursors of branched fatty alcohols in vivo.
The strain MG1655(DE3) ΔfadE::FRT ΔfabH::cat/pDG6 was constructed as follows:
A region of the E. coli fabH gene described in Lai, et al., 2003, J. Biol. Chem. 278(51): 59494, was replaced by an antibiotic resistance gene. This deletion was performed in a strain that was complemented for fabH by the plasmid pDG6 carrying the B. subtilis fabH1 gene.
Initially, the pDG2 plasmid was constructed. The pCDFDuet-1 vector was purchased from Novagen/EMD Biosciences. The vector carries the CloDF13 replicon, lacI gene and streptomycin/spectinomycin resistance gene (aadA).
The C-terminal portion of the plsX gene, which contains an internal promoter for the downstream fabH gene, was amplified from E. coli MG1655 genomic DNA using primers 5′-TGAATTCCATGGCGCAACTCACTCTTCTTTTAGTCG-3′ (SEQ ID NO:168) and 5′-CAGTACCTCGAGTCTTCGTATACATATGCGCT CAGTCAC-3′ (SEQ ID NO:169). These primers introduced NcoI and XhoI restriction sites near the ends, as well as an internal NdeI site.
Both the plsX insert and pCDFDuet-1 vector were digested with restriction enzymes NcoI and XhoI. The cut vector was treated with Antarctic phosphatase. The insert was ligated into the vector and transformed into chemically competent TOP10 cells. Clones were screened by DNA sequencing. See, FIG. 6A.
Then a pDG6 plasmid was constructed using the pDG2 plasmid. The fabH1gene from Bacillus subtilis strain 168 was amplified from plasmid pLS9-114 (see, FIG. 6K) using primers 5′-CCTTGGGGCATATGAAAGCTG-3′ (SEQ ID NO:170) and 5′-TTTAGTCATCTCGAGTGCACCTCACCTTT-3′ (SEQ ID NO:171). These primers introduced or included NdeI and XhoI restriction sites.
Both the fabH1 insert and pDG2 vector were digested with restriction enzymes NdeI and XhoI. The cut vector was treated with Antarctic phosphatase. The insert was ligated into the vector and transformed into chemically competent TOP10 cells. Clones were screened by DNA sequencing. See, FIG. 6B and FIG. 6C.
Then, the cat chloramphenicol resistance gene was amplified from template plasmid pKD3 using primers 5′-GCCACATTGCCGCGCCAAACGAAACC GTTTCAACCATGGCATATGAATATCCTCCTTAGTTCCTATTCCG-3′ (SEQ ID NO: 172) and 5′-CGCCCCAGATTTCACGTATTGATCGGCTACGCTTAATGCAT GTGTAGGCTGGAGCTGCTTC-3′ (SEQ ID NO:173) which added 50 bp nucleotide ends that are homologous to the E. coli fabH gene. This linear PCR product was used to inactivate the E. coli fabH gene.
Strain MG1655(DE3) ΔfadE::FRT was first transformed with plasmid pKD46 encoding the lambda red recombinase genes. MG1655(DE3) ΔfadE::FRT/pKD46 was then transformed with plasmid pDG6. Finally, MG1655(DE3) ΔfadE::FRT/pKD46+pDG6 was induced for expression of the recombinase genes by addition of 10 mM arabinose and transformed with the linear PCR product as described in Datsenko et al. (supra). Colonies were selected on LB plates containing 30 μg/mL chloramphenicol and screened using colony PCR with primers 5′-TTGACACGTC TAACCCTGGC-3′ (SEQ ID NO:174) and 5′-CTGTCCAGGGAACACAAATG C-3′ (SEQ ID NO:175).
A number of other constructs comprising pDG7, and pDG8 were also constructed following the approach as above. The plasmids are prepared as follows.
The plasmid pDG7 was prepared from pDG2 with B. subtilis fabH2 insert. The fabH2 gene from Bacillus subtilis strain 168 was amplified from plasmid pLS9-111 (see, FIG. 6J) using primers 5′-TTGTGTCGCCCTTTCGCTG-3′(SEQ ID NO:176) and 5′-CTTACGTACGTACTCGAGTGACGC-3′ (SEQ ID NO:177). These primers introduced or included NdeI and XhoI restriction sites.
Both the fabH2 insert and pDG2 vector were digested with restriction enzymes NdeI and XhoI. The cut vector was treated with Antarctic phosphatase. The insert was ligated into the vector and transformed into chemically competent TOP10 cells. Clones were screened by DNA sequencing. See, FIG. 6D and FIG. 6E.
The plasmid pDG8 was prepared from pDG2 with S. coelicolor fabH insert. The fabH gene from Streptomyces coelicolor was amplified from plasmid pLS9-115 (see, FIG. 6L) using primers 5′-AAGTGGGGCATATGTCTAAGATC-3′ (SEQ ID NO:178) and 5′-GTGATCCGGCTCGAGGTGGTTAC-3′ (SEQ ID NO:179). These primers introduced or included NdeI and XhoI restriction sites.
Both the fabH insert and pDG2 vector were digested with restriction enzymes NdeI and XhoI. The cut vector was treated with Antarctic phosphatase. The insert was ligated into the vector and transformed into chemically competent TOP10 cells. Clones were screened by DNA sequencing. See, FIG. 6F and FIG. 6G.
The plasmid pDG10 was prepared using pCR-Blunt vector, which was purchased from Invitrogen, with C. acetobutylicum ptb_buk operon insert, wherein the ptb part represents the gene encoding C. acetobutylicum phosphotransbutyrylase (GenBank Accession AAA75486.1, SEQ ID NO:156), and the buk part represents the gene encoding C. acetobutylicum butyrate kinase (GenBank Accession JN0795, SEQ ID NO:157). The buk_ptb operon was amplified from Clostridium acetobutylicum genomic DNA (ATCC 824) using primers 5′-CTTAACTTCATGTGAAAAGTTTGT-3′ (SEQ ID NO:180) and 5′-ACAATACCCATGTTTATAGGGCAA-3′ (SEQ ID NO:181). The PCR product was ligated into the pCR Blunt vector following the manufacturer's instructions. Colonies were verified by DNA sequencing. See, FIG. 6H and FIG. 6I.
E. coli strains were transformed with pDG10, and OP-180 plasmid comprising E. coli thioesterase gene tesA under the control of the Ptrc promoter and independently also one of the pDG6, pDG7 and pDG8 plasmids as described above.
These strains were fed branched molecules isobutyrate, which resulted in iso-C14:0 and iso-C16:0 branched acyl-CoA precursors. Independently they were fed branched molecule isovalerate, which resulted in iso-C13:0 and iso-C15:0 branched acyl-CoA precursors. See, FIG. 4B. These precursors can then be incorporated into the branched fatty alcohol pathways as described herein and depicted in FIG. 1A and FIG. 1B.
Other Embodiments
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

## Claims

1. A method of making a surfactant composition using branched chain fatty alcohols produced in a recombinant host cell, the method comprising,
(a) obtaining a recombinant host cell genetically modified to comprise (i) a polynucleotide encoding a branched chain alpha-keto acid dehydrogenase (BKD) complex, (ii) a polynucleotide encoding a polypeptide having beta-ketoacyl-ACP synthase (FabH) activity that utilizes a branched acyl-CoA molecule as a substrate, and (iii) a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide selected from an acyl-ACP reductase (AAR) that utilizes a branched fatty acyl-ACP as a substrate, and a carboxylic acid reductase (CAR) that utilizes a branched fatty acid as a substrate when a thioesterase is present;
(b) culturing the recombinant host cell in the presence of a carbon source under conditions effective to express the polynucleotides and produce branched chain fatty alcohols that are secreted into the extracellular environment of the host cell;
(c) collecting the branched chain fatty alcohols; and
(d) blending the branched chain fatty alcohols to make a surfactant composition.

2. The method of claim 1, further comprising a polypeptide having fatty alcohol biosynthesis activity.

3. The method of claim 2, wherein said polypeptide having fatty alcohol biosynthesis activity is an alcohol dehydrogenase (EC 1.1.1.1).

4. The method of claim 1, wherein the branched chain fatty alcohols include one or more of saturated or unsaturated C 12, C 14 and C 16 fatty alcohols.