Patent Grant
10392606
The present disclosure relates to enzymes, mutants and chimeras thereof, fatty acid synthesis, nucleic acids, proteins and host cells and organisms.
De novo fatty acid biosynthesis can be considered an iterative “polymerization” process, commonly primed with the acetyl moiety from acetyl-CoA and with iterative chain extension occurring by reaction with malonyl-acyl carrier protein (ACP). In most organisms this process optimally produces 16- and 18-carbon (C16 and C18) fatty acids. The enzyme that determines fatty acid chain length is acyl-ACP thioesterase (TE). This enzyme catalyzes the terminal reaction of fatty acid biosynthesis, acyl-ACP thioester bond hydrolysis (i.e., the hydrolysis of the thioester bond between the acyl chain and the sulfhydryl group of the phosphopantetheine prosthetic group of ACP), to release a free fatty acid and ACP. This reaction terminates acyl-chain elongation of fatty acid biosynthesis and, therefore, determines fatty acid chain length. It is also the biochemical determinant of the fatty acid composition of storage lipids in plant seeds.
In discrete phyla and/or tissues of specific organisms (primarily higher plant seeds), thioester hydrolysis optimally produces medium-chain (C8-C14) fatty acids (MCFAs), which have wide industrial applications (e.g., producing detergents, lubricants, cosmetics, and pharmaceuticals) (Dehesh et al., Plant Physiol. 110: 203-210 (1996)). TEs that specifically hydrolyze medium-chain acyl-ACP substrates have been studied widely (Dehesh et al. (1996), supra; Voelker et al., Science 257: 72-74 (1992)); and Yuan et al., PNAS USA 92: 10639-10643 (1995)). Short-chain fatty acids (SCFAs; e.g., butanoic acid and hexanoic acid) have more recently gained importance as potential bio-renewable chemicals that could be derived from the fatty acid biosynthesis pathway (Nikolau et al., Plant J. 54: 536-545 (2008)). As a critical acyl chain termination enzyme, acyl-ACP TEs with desired substrate specificities are, therefore, important for engineering this pathway.
To date, dozens of acyl-ACP TEs have been functionally characterized and sorted into two classes, FatA and FatB (Jones et al., Plant Cell 7: 359-371 (1995)). FatA-class TEs act on long-chain acyl-ACPs, preferentially on oleoyl-ACP (Jones et al. (1995), supra; Hawkins et al., Plant J. 13: 743-752 (1998); Serrano-Vega et al., Planta 221: 868-880 (2005); and Sanchez-Garcia et al., Phytochemistry 71: 860-869 (2010)), while FatB-class TEs preferably hydrolyze acyl-ACPs with saturated fatty acyl chains (Jones et al. (1995), supra). The archetypical FatB-class TE was isolated from the developing seeds of California bay (Umbellularia californica). This enzyme is specific for 12:0-ACP, and it plays a critical role in MCFA production (Voelker et al. (1992), supra; and Pollard et al., Arch Biochem. Biophys. 284: 306-312 (1991)). This discovery spurred isolation of additional MCFA-specific TEs from Cuphea (Dehesh et al. (1996), supra; Dehesh et al. Plant J. 9: 167-172 (1996); and Leonard et al., Plant Mol. Biol. 34: 669-679 (1997)), Arabidopsis thaliana (Dormann et al., Arch Biochem. Biophys. 316: 612-618 (1995)), Myristica fragrans (nutmeg) (Voelker et al., Plant Physiol. 114: 669-677 (1997)), and Ulmus americana (elm) (Voelker et al. (1997), supra).
Recently, TEs obtained from public databases were classified into 23 families based on sequence and three-dimensional structure similarity (Cantu et al., Protein Sci. 19: 1281-1295 (2010)). These TEs were defined as enzymes that can hydrolyze any thioester bond irrespective of the chemical nature of the carboxylic acid and thiol molecules that constitute the substrates of these enzymes. The TE sequences are collected in the constantly updated ThYme database (on the worldwide web at enzyme.cbirciastate.edu; Cantu et al., Nucleic Acids Res. 39: D342-346 (2011), which is hereby incorporated by reference). Of these 23 families, Family TE14 contains plant and bacterial acyl-ACP TEs involved in Type II fatty acid synthesis, the reactions of which are catalyzed by discrete mono-functional enzymes. Family TE14 contained 360 unique sequences as of late 2010, but only ˜7% of these sequences had been functionally characterized, and all of those were FatA and FatB TEs from higher plants. The remaining ˜220 bacterial acyl-ACP TEs were mostly generated from genomic sequencing projects and had not been functionally characterized.
Alteration of the substrate specificity of plant TEs has been described by Yuan et al. (U.S. Pat. Nos. 5,955,329 and 6,150,512, which are incorporated herein by reference for their teachings regarding same) and Roessler et al. (U.S. Pat. App. Pub. No. 2011/0020883, which is hereby incorporated by reference for its teachings regarding same). Yuan et al. identifies the C-terminal two-thirds portion of plant TEs as desirable for modification. Roessler et al. discloses a plant acyl-ACP thioesterase of a specified sequence (sequence identification no. 29) in which amino acid 174, alone or in further combination with amino acid 103, is mutated.
In view of the foregoing, the present disclosure seeks to provide methods of using acyl-ACP TE and mutants and chimeras thereof, in particular bacterial and plant acyl-ACP TE and mutants and chimeras thereof, to alter substrate specificity and/or alter activity (e.g., increase production of fatty acids) in a host cell or organism. These and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.
A method of increasing production of fatty acids, such as short-chain fatty acids (e.g., fatty acids having less than about six carbons) and/or fatty acids having from about six carbons to about 12 carbons, such as from about 10 carbons to about 12 carbons (e.g., fatty acids having less than about 10 carbons or fatty acids having less than about 12 carbons) in a host cell or organism is provided. The method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding an acyl-acyl carrier protein (ACP) thioesterase (TE) from Bryantella formatexigens.
Another method of increasing production of fatty acids, such as short-chain fatty acids (e.g., fatty acids having less than about six carbons) and/or fatty acids having from about six carbons to about 12 carbons, such as from about 10 carbons to about 12 carbons (e.g., fatty acids having less than about 10 carbons or fatty acids having less than about 12 carbons) in a host cell or organism is also provided. The method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE derived from wild-type Bryantella formatexigens acyl-ACP TE, wherein the mutant acyl-ACP TE produces more fatty acids, such as short-chain fatty acids, in the host cell or organism that the corresponding wild-type acyl-ACP TE.
Also provided is a method of making a mutant Bryantella formatexigens acyl-ACP TE. The method comprises making a mutant Bryantella formatexigens acyl-ACP TE comprising two or more amino acid mutations comprising N169Y and S222I.
An isolated or purified nucleic acid molecule is also provided. The nucleic acid molecule comprises a nucleotide sequence encoding a mutant acyl-ACP TE, which is derived from wild-type Bryantella formatexigens acyl-ACP TE, comprises two or more amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type Bryantella formatexigens acyl-ACP TE. The isolated or purified nucleic acid molecule can be a vector.
Also provided is a host cell or organism. The host cell or organism comprises the above-described nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE.
Further provided is an isolated or purified mutant acyl-ACP TE. The mutant acyl-ACP TE is derived from wild-type Bryantella formatexigens acyl-ACP TE, comprises two or more amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type Bryantella formatexigens acyl-ACP TE.
A method of making a chimeric Cuphea viscosissima acyl-ACP TE gene is also provided. The method comprises replacing a segment of a wild-type Cuphea viscosissima acyl-ACP TE with a segment of another acyl-ACP TE.
Further provided is another isolated or purified nucleic acid molecule. The nucleic acid molecule comprises a nucleotide sequence encoding a chimeric Cuphea viscosissima acyl-ACP TE gene, which comprises a segment of another acyl-ACP TE gene. The isolated or purified nucleic acid molecule can be a vector.
Still further provided is another host cell or organism. The host cell or organism comprises the above-described isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a chimeric Cuphea viscosissima acyl-ACP TE gene.
Even still further provided is an isolated or purified chimeric Cuphea viscosissima acyl-ACP TE. The chimera comprises a segment of another acyl-ACP TE.
A method of altering the specificity of a plant acyl-ACP TE for at least one of its substrates is also provided. The method comprises introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or the region corresponding to amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The method can comprise mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The method can further comprise mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The method can further comprise altering the level of activity of the plant acyl-ACP TE by a method comprising mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
In view of the foregoing, a method of altering the level of activity of a plant acyl-ACP TE and the specificity of the plant acyl-ACP TE for at least one of its substrates is also provided. The method comprises (i) mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The method can comprise mutating at least one amino acid corresponding to an amino selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The method can further comprise mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Yet another isolated or purified nucleic acid molecule is provided. The isolated or purified nucleic acid molecule comprises a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The isolated or purified nucleic acid molecule can be a vector. The encoded mutant plant acyl-ACP TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The encoded mutant plant acyl-ACP TE can further comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The encoded mutant plant acyl-ACP TE can further comprise a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Still yet another isolated or purified nucleic acid molecule is provided. The isolated or purified nucleic acid molecule comprises a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises (i) a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The encoded mutant plant acyl-ACP TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The encoded mutant plant acyl-ACP TE can further comprise a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Another host cell or organism is provided. The host cell or organism comprises the above-described isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
An isolated or purified mutant plant acyl-ACP TE is also provided. The isolated or purified mutant plant acyl-ACP TE comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Yet another isolated or purified mutant plant acyl-ACP TE is provided. The isolated or purified mutant plant acyl-ACP TE comprises (i) a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
The present disclosure is predicated, at least in part, on the discovery that expression of an acyl-acyl carrier protein (ACP) thioesterase (TE) from Bryantella formatexigens (DSM 14469, EET61113.1, ZP_05345975.3, C6LDQ9; Wolin et al., Appl. Environ. Microbiol. 69(10): 6321-6326 (October 2003); nucleotide sequence is SEQ ID NO: 23; amino acid sequence is SEQ ID NO: 24) in a host cell or organism, such as Escherichia coli, results in an increase in the production of short-chain fatty acids in the host cell or organism. In this regard, it has been surprisingly and unexpectedly discovered that the introduction of two or more point mutations in the acyl-ACP TE of B. formatexigens has a synergistic effect on short-chain activity in the host cell or organism. The present disclosure is further predicated on the discovery that the substrate specificity and activity (e.g., total amount of fatty acids produced) of an acyl-ACP TE, such as a plant acyl-ACP TE, can be affected by introducing a substrate specificity-altering mutation in the region corresponding to amino acids 118-167, such as from about amino acid 118 to about amino acid 167, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 and/or by introducing a substrate specificity-altering mutation in the region corresponding to amino acids 73-85, such as from about amino acid 73 to about amino acid 85, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2. The materials and methods have application for biofuels, industrial lubricants, food oils, and the like.
In view of the foregoing, a method of increasing production of fatty acids, such as short-chain fatty acids (e.g., fatty acids having less than about six carbons) and/or fatty acids having from about six to about 12 carbon atoms (C6-C12), such as fatty acids having from about 10 to about 12 carbon atoms (C10-C12), in a host cell or organism is provided. Thus, the method can be used to increase production of fatty acids having less than about 10 carbon atoms or fatty acids having less than about 12 carbon atoms. The method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding an acyl-ACP TE, such as an acyl-ACP TE from
Another method of increasing production of fatty acids, such as short-chain fatty acids (e.g., fatty acids having less than about six carbons) and/or fatty acids having from about six to about 12 carbon atoms (C6-C12), such as fatty acids having from about 10 to about 12 carbon atoms (C10-C12), in a host cell or organism is also provided. The method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE derived from a wild-type acyl-ACP TE, such as an acyl-ACP TE from
A method of making a mutant Bryantella formatexigens acyl-ACP TE is also provided. The method comprises making a mutant Bryantella formatexigens acyl-ACP TE comprising two or more amino acid mutations comprising N169Y and S222I. Preferably, and even desirably, the mutant Bryantella formatexigens acyl-ACP TE has increased thioesterase activity compared to a corresponding wild-type Bryantella formatexigens acyl-ACP TE. Mutant acyl-ACP TEs can be derived from such wild-type acyl-ACP TEs in accordance with methods known in the art and exemplified herein.
Also provided is a method of making a chimeric Cuphea viscosissima acyl-ACP TE. Any suitable method of making a chimera as known in the art and exemplified herein can be used. The method can comprise replacing a segment of a wild-type Cuphea viscosissima acyl-ACP TE with a segment of another acyl-ACP TE. Examples of wild-type Cuphea viscosissima acyl-ACP TEs include those encoded by the FatB1 gene (designated CvFatB1) and the FatB2 gene (designated CvFatB2). Any suitable acyl-ACP TE (see, e.g.,
Acetivibrio cellulolyticus CD2
Acetobacterium woodii (strain ATCC
Akkermansia muciniphila (strain ATCC
Alistipes finegoldii DSM 17242
Alistipes shahii WAL 8301
Alistipes sp. HGB5
Alkaliphilus oremlandii (strain OhlLAs)
Anaeromyxobacter dehalogenans (strain
Anaeromyxobacter sp. (strain Fw109-5)
Anaeromyxobacter sp. (strain K)
Anaerophaga sp. HS1
Anaerophaga thermohalophila DSM
Anaerostipes sp. 3_2_56FAA
Arcobacter nitrofigilis (strain ATCC
Arcobacter sp. L.
Atopobium parvulum (strain ATCC
Bacillus coagulans (strain 2-6)
Bacteroides clarus YIT 12056
Bacteroides coprosuis DSM 18011
Bacteroides dorei 5_1_36/D4
Bacteroides eggerthii 1_2_48FAA
Bacteroides faecis MAJ27
Bacteroides fluxus YIT 12057
Bacteroides fragilis (strain YCH46)
Bacteroides fragilis 3_1_12
Bacteroides helcogenes (strain ATCC
Bacteroides ovatus SD CC 2a
Bacteroides salanitronis (strain DSM
Bacteroides sp. 1_1_14
Bacteroides sp. 20_3
Bacteroides sp. 3_1_19
Bacteroides sp. 3_1_23
Bacteroides sp. 3_1_33FAA
Bacteroides sp. 3_1_40A
Bacteroides sp. 4_1_36
Bacteroides sp. D22
Bacteroides thetaiotaomicron (strain
Bacteroides vulgates (strain ATCC
Bacteroides xylanisolvens SD CC 1b.
Bacteorides xylanisolvens XB1A
Bilophila wadsworthia 3_1_6
Butyrivibrio crossotus DSM 2876
Butyrivibrio fibrisolvens
Butyrivibrio proteoclasticus (strain
Clostridium proteoclasticum)
Calditerrivibrio nitroreducens (strain
Caldithrix abyssi DSM 13497
Capnocytophaga gingivalis ATCC 33624
Capnocytophaga ochracea (strain ATCC
Capnocytophaga ochracea F0287
Capnocytophaga sp. oral taxon 329 str.
Capnocytophaga sp. oral taxon 329 str.
Capnocytophaga sp. oral taxon 338 str.
Capnocytophaga sputigena ATCC 33612
Carnobacterium maltaromaticum ATCC
Carnobacterium sp. (strain 17-4)
Cellulosilyticum lentocellum (strain
Cellulosilyticum lentocellum (strain
Clostridium acetobutylicum (strain
Clostridium beijerinckii (strain ATCC
acetobutylicum)
Clostridium beijerinckii (strain ATCC
acetobutylicum)
Clostridium botulinum (strain 657/Type
Clostridium botulinum (strain Alaska
Clostridium botulinum (strain ATCC
Clostridium botulinum (strain Eklund
Clostridium botulinum (strain H04402
Clostridium botulinum (strain
Clostridium botulinum (strain Loch
Clostridium botulinum BKT015925
Clostridium botulinum C str. Eklund
Clostridium botulinum C str. Stockholm
Clostridium botulinum D str. 1873
Clostridium botulinum E1 str. ‘BoNT E
Clostridium butyricum E4 str. BoNT E
Clostridium carboxidivorans P7
Clostridium cellulolyticum (strain ATCC
Clostridium cellulovorans (strain ATCC
Clostridium hahtewayi DSM 13479
Clostridium novyi (strain NT)
Clostridium papyrosolvens DSM 2782
Clostridium pelfringens (strain ATCC
Clostridium pelfringens (strain ATCC
Clostridium pelfringens (strain
Clostridium pelfringens CPE str. F4969
Clostridium pelfringens CPE str. F4969
Clostridium pelfringens F262
Clostridium pelfringens F262
Clostridium phytofermentans (strain
Clostridium saccharolyticum (strain
Clostridium saccharolyticum
Clostridium sp. 7_2_43FAA
Clostridium sp. 7_2_43FAA
Clostridium sp. BNL1100
Clostridium sp. DL-VIII
Clostridium sp. M62/1
Clostridium sporogenes PA 3679
Clostridium tetani (strain
Clostridium thermocellum (strain DSM
Coprococcus catus GD/7
Coprococcus sp. ART55/1
Cryptobacterium curtum (strain ATCC
Deferribacter desulfuricans (strain DSM
Denitrovibrio acetiphilus (strain DSM
Desulfatibacillum alkenivorans (strain
Desulfitobacterium dehalgenans ATCC
Desulfitobacterium hafniense (strain
Desulfitobacterium
metallireducens DSM
Desulfobacterium autotrophicum (strain
Desulfococcus oleovorans (strain DSM
Desulfohalobium retbaense (strain DSM
Desulfomicrobium baculatum (strain
baculatus)
Desulfonatronospira thiodismutans
Desulfosporosinus meridiei DSM 13257
Desulfosporosinus orientis (strain ATCC
Desulfosporosinus youngiae DSM 17734
Desulfotomaculum carboxydivorans
Desulfotomaculum nigrificans DSM 574
Desulfovibrio aespoeensis (strain ATCC
Desulfovibrio africanus str. Walvis Bay
Desulfovibrio desulfuricans (strain G20)
Desulfovibrio desulfuricans ND132
Desulfovibrio salexigens (strain ATCC
Desulfovibrio sp. A2
Desulfovibrio vulgaris (strain Miyazaki
Dethiosulfovibrio peptidovorans DSM
Elusimicrobium
minutum (strain Pei191)
Enterococcus casseliflavus ATCC 12755
Enterococcus casseliflavus EC10
Enterococcus casseliflavus EC20
Enterococcus faecalis (strain 62)
Enterococcus faecalis (strain ATCC
Enterococcus faecalis ATCC 29200
Enterococcus faecalis DAPTO 512
Enterococcus faecalis Merz96
Enterococcus faecalis T11
Enterococcus faecalis T2
Enterococcus faecalis T8
Enterococcus faecalis TUSoD Ef11
Enterococcus faecalis TX0012
Enterococcus faecalis TX0104
Enterococcus faecalis TX0309B
Enterococcus faecalis TX1467
Enterococcus faecalis TX2141
Enterococcus faecalis TX4000
Enterococcus faecium (strain Aus0004)
Enterococcus faecium Com15
Enterococcus faecium E1636
Enterococcus faecium E1679
Enterococcus faecium E980
Enterococcus faecium PC4.1
Enterococcus faecium TX0133a01
Enterococcus faecium TX1330
Enterococcus gallinarum EG2
Enterococcus italicus DSM 15952
Ethanoligenens harbinense (strain DSM
Eubacterium cellulosolvens 6
Eubacterium limosum (strain KIST612)
Eubacterium rectale DSM 17629
Eubacterium rectal M104/1
Eubacterium saburreum DSM 3986
Eubacterium siraeum 70/3
Eubacterium siraeum V10Sc8a
Faecalibacterium prausnitzii L2-6
Fibrella aestuarina BUZ 2
Fibrisoma limi BUZ 3
Fibrobacter succinogenes (strain ATCC
Finegoldia magna (strain ATCC 29328)
Finegoldia magna
Flavobacterium columnare (strain ATCC
Flavobacterium frigoris PS1
Flavobacterium johnsoniae (Strain
Cytophaga johnsonae
Flavonifractor plautii ATCC 29863
Flexistipes sinusarabici (strain DSM
Fructobacillus fructosus KCTC 3544
Geobacillus sp. (strain Y412MC10)
Geobacter lovleyi (Strain ATCC BAA-
Geobacter metallireducens (strain GS-
Geobacter sp. (strain FRC-32)
Granulicatella adiacens ATCC 49175
Granulicatella elegans ATCC 700633
Haliscomenobacter hydrossis (strain
Haloplasma contractile SSD-17B
Jonquetella anthropi DSM 22815
Jonquetella anthropi E3_33 E1
Lachnospiraceae bacterium 5_1_63FAA
Lachnospiraceae bacterium oral taxon
Lactobacillus acidipiscis KCTC 13900
Lactobacillus acidophilus (strain 30SC)
Lactobacillus aciophilus (strain ATCC
Lactobacillus acidophilus ATCC 4796
Lactobacillus amylolyticus DSM 11664
Lactobacillus amylovorus (strain GRL
Lactobacillus animalis KCTC 3501
Lactobacillus brevis (strain ATCC
Lactobacillus brevis subsp. gravesensis
Lactobacillus buchneri (strain NRRL B-
Lactabacillus buchneri ATCC 11577
Lactobacillus casei (strain ATCC 334)
Lactobacillus coleohominis 101-4-CHN
Lactobacillus crispatus (strain ST1)
Lactobacillus crispatus 125-2-CHN
Lactobacillus crispatus CTV-05
Lactobacillus crispatus MV-1A-US
Lactobacillus delbrueckii subsp.
bulgaricus (strain 2038)
Lactobacillus delbrueckii subsp.
bulgaricus (strain ATCC BAA-365)
Lactobacillus delbrueckii subsp.
bulgaricus (strain ND02)
Lactobacillus delbrueckii subsp.
bulgaricus PB2003/044-T3-4
Lactobacillus delbrueckii subsp. lactis
Lactobacillus farciminis KCTC 3681
Lactobacillus fermentum (strain CECT
Lactobacillus fructivorans KCTC 3543
Lactobacillus gasseri (strain ATCC
Lactobacillus gasseri 202-4
Lactobacillus gasseri 224-1
Lactobacillus gasseri JV-V03
Lactobacillus gastricus P53
Lactobacillus helveticus (strain H10)
Lactobacillus helveticus MTCC 5463
Lactobacillus hilgardii ATCC 8290
Lactobacillus iners AB-1
Lactobacillus iners ATCC 55195
Lactobacillus iners LactinV 09V1-c
Lactobacillus iners LEAF 3008A-a
Lactobacillus iners SPIN 1401G
Lactobacillus iners SPIN 2503V10-D
Lactobacillus iners UPII 143-D
Lactobacillus iners UPII 60-B
Lactobacillus jensenii 208-1
Lactobacillus johnsonii DPC 6026
Lactobacillus johnsonii pf01
Lactobacillus kefiranofaciens (strain
Lactobacillus kisonensis F0435
Lactobacillus malefermentans KCTC
Lactobacillus mali KCTC 3596 = DSM
Lactobacillus mucosae LM1
Lactobacillus oris F0423
Lactobacillus oris PB013-T2-3
Lactobacillus paracasei subsp. paracasei
Lactobacillus parafarraginis F0439
Lactobacillus plantarum (strain ATCC
Lactobacillus reuteri (strain ATCC
Lactobacillus reuteri 100-23
Lactobacillus rhamnosus (strain ATCC
Lactobacillus rhamnosus (strain Lc 705)
Lactobacillus ruminis (strain ATCC
Lactobacillus ruminis SPM0211
Lactobacillus salivarius (strain CECT
Lactobacillus salivarius (strain UCC118)
Lactobacillus salivarius ACS-116-V-
Lactobacillus salvarius GJ-24
Lactobacillus salivarius SMXD51
Lactobacillus vaginalis ATCC 49540
Lactobacillus versmoldensis KCTC 3814
Lactobacillus zeae KCTC 3804
Lactococcus garvieae IPLA 31405
Lactococcus garvieae (strain Lg2)
Lactococcus lactis subsp. cremoris
Lactococcus lactis subsp. cremoris A76
Lactococcus lactis subsp. lactis (strain
Lactococcus lactis subsp. lactis IO-1
Leuconostoc citreum (strain KM20)
Leuconostoc citreum LBAE C10
Leuconostoc citreum LBAE C11
Leuconostoc fallax KCTC 3537
Leuconostoc mesenteroides subsp.
mesenteroides (strain ATCC 8293/NCDO
Leuconostoc pseudomesenteroides KCTC
Leuconostoc pseudomesenteroides KCTC
Marvinbryantia formatexigens DSM
Melissococcus plutonius (strain ATCC
Melissococcus plutonius (strain
Mesotoga prima MesG1.Ag.4.2
Microscilla marina ATCC 23134
Moorella thermoacetica (strain ATCC
Myroides odoratus DSM 2801
Odoribacter splanchnicus (strain ATCC
splanchnicus)
Oenococcus kitaharae DSM 17330
Olsenella uli (strain ATCC 49627/DSM
Opitutus terrae (strain DSM
Ornithinibacillus scapharcae TW25
Paenibacillus dendritiformis C454
Paenibacillus lactis 154
Paenibacillus sp. HGF5
Paenibacillus vortex V453
Paludibacter propionici genes (strain
Parabacteroides distasonis (strain ATCC
Parabacteroides sp. D13
Paraprevotella clara YIT 11840
Paraprevotella xylaniphila YIT 11841
Pediococcus acidilactici DSM 20284
Pediococcus acidilactici MA18/5M
Pediococcus pentosaceus (strain ATCC
Pelobacter propionicus (strain DSM
Peptoniphilus harei ACS-146-V-Sch2b
Prevotella bivia JCVIHMP010
Prevotella buccae ATCC 33574
Prevotella buccalis ATCC 35310
Prevotella dentalis DSM 3688
Prevotella dentalis DSM 3688
Prevotella denticola (strain F0289)
Prevotella denticola CRIS 18C-A
Prevotella disiens FB035-09AN
Prevotella intermedia 17
Prevotela marshii DSM 16973
Prevotella melaninogenica (strain ATCC
melaninogenicus)
Prevotella multiformis DSM 16608
Prevotella multisaccharivorax DSM
Prevotella nigrescens ATCC 33563
Prevotella orails ATCC 33269
Prevotella oris C735
Prevotella ruminicola (strain ATCC
Prevotella salivae DSM 15606
Prevotella sp. oral taxon 306 str. F0472
Prevotella stercorea DSM 18206
Pseudoramibacter alactolyticus ATCC
Rhodothermus marinus (strain ATCC
obamensis)
Rhodothermuss marinus SG0.5JP17-172
Roseburia hominis (strain DSM
Roseburia intestinalis L1-82
Roseburia intestinalis XB6B4
Ruminococcus albus (strain ATCC
Ruminococcus obeum A2-162
Ruminococcus sp. SR1/5
Ruminococcus torques L2-14
Salinibacter ruber (strain DSM
Salinibacter ruber (strain M8)
Sphaerochaeta pleomorpha (strain ATCC
Sphingobacterium spiritovorum ATCC
Sphingobacterium spiritivorum ATCC
Spirochaeta africana DSM 8902
Sprochaeta caldaria (strain ATCC
Sprochaeta caldaria (strain ATCC
Spirochaeta coccoides (strain ATCC
Spirochaeta coccoides (strain ATCC
Spirochaeta sinaragdinae (strain DSM
Spirochaeta sinaragdinae (strain DSM
Spirochaeta thermophila (strain ATCC
Spirochaeta thermophila DSM 6578
Spirosoina linguale (strain ATCC
Streptococcus anginosus 1_2_62CV
Streptococcus anginosus CGUG 39159
Streptococcus anginosus F0211
Streptococcus anginosus SK52 = DSM
Streptococcus australis ATCC 700641
Streptococcus canis FSL Z3-227
Streptococcus constellatus subsp.
constellatus SK53
Streptococcus constellatus subsp.
pharyngis SK1060 = CCUG 46377
Streptococcus criceti HS-6
Streptococcus cristatus ATCC 51100
Streptococcus downei F0415
Streptococcus dysgalactiae subsp.
equisinalis (strain ATCC 12394/D166B)
Streptococcus dysgalactiae subsp.
equisinalis (strain GGS_124)
Streptococcus dysgalactiae subsp.
equismilis SK1249
Streptococcus equi subsp. zooepideinicus
Streptococcus equi subsp. zooepideinicus
Streptococcus equines ATCC 9812
Streptococcus gallolyticus subsp.
gallolyticus TX20005
Streptococcus ictaluri 707-05
Streptococcus infantarius (strain CJ18)
Streptococcus infantis ATCC 700779
Streptococcus infantis SK1076
Streptococcus infantis SK1302
Streptococcus infantis SK970
Streptococcus infantis X
Streptococcus intermedius SK54
Streptococcus macacae NCTC 11558
Streptococcus macedonicus (strain ACA-
Streptococcus mitts (strain B6)
Streptococcus mitts ATCC 6249
Streptococcus mitts by. 2 str. F0392
Streptococcus mitts by. 2 str. SK95
Streptococcus mitts NCTC 12261
Streptococcus mitts SK1073
Streptococcus mitts SK1080
Streptococcus mitts SK569
Streptococcus mitts SK575
Streptococcus mitts SK579
Streptococcus mitts SK597
Streptococcus mitts SK616
Streptococcus oralis SK10
Streptococcus oralis SK100
Streptococcus oralis SK1074
Streptococcus oralis SK255
Streptococcus oralis SK313
Streptococcus oralis SK610
Streptococcus parasanguinis F0449
Streptococcus parasanguinis (strain
Streptococcus parasanguinis ATCC 903
Streptococcus parasanguinis F0405
Streptococcus parasanguinis SK236
Streptococcus parauberis (strain KCTC
Streptococcus parauberis NCFD 2020
Streptococcus peroris ATCC 700780
Streptococcus pneumoniae (strain 670-
Streptococcus pneumoniae (strain 70585)
Streptococcus pneumoniae (strain
Streptococcus pneumoniae GA05245
Streptococcus pneumoniae GA11663
Streptococcus pneumoniae GA13637
Streptococcus pneumoniae GA40028
Streptococcus pneumoniae GA40563
Streptococcus pneumoniae GA41688
Streptococcus pneumoniae GA47373
Streptococcus pneumoniae GA47439
Streptococcus pneumoniae GA47461
Streptococcus pneumoniae GA47522
Streptococcus pneumoniae GA47778
Streptococcus pneumoniae GA49194
Streptococcus pneumoniae GA49542
Streptococcus porcinus str Jelinkova 176
Streptococcus pseudopneumoniae (strain
Streptococcus pseudopneumoniae SK674
Streptococcus pseudoporcinus LQ 940-
Streptococcus pyogenes HKU
Streptococcus pyogenes Alab49
Streptococcus pyogenes ATCC 10782
Streptococcus pyogenes MGAS1882
Streptococcus pyogenes serotype M1
Streptococcus pyogenes serotype M12
Streptococcus pyogenes serotype M2
Streptococcus pyogenes serotype M28
Streptococcus pyogenes serotype M4
Streptococcus pyogenes serotype M6
Streptococcus salivarius (strain 57.I)
Streptococcus salivarius SK126
Streptococcus sanguinis ATCC 49296
Streptococcus sanguinis SK1056
Streptococcus sanguinis SK1057
Streptococcus sanguinis SK1058
Streptococcus sanguinis SK1087
Streptococcus sanguinis SK115
Streptococcus sanguinis SK150
Streptococcus sanguinis SK160
Streptococcus sanguinis SK330
Streptococcus sanguinis SK340
Streptococcus sanguinis SK353
Streptococcus sanguinis SK355
Streptococcus sanguinis SK408
Streptococcus sanguinis SK49
Streptococcus sanguinis SK678
Streptococcus sanguinis SK72
Streptococcus sp. SK140
Streptococcus sp. SK643
Streptococcus sp. C300
Streptococcus sp. M143
Streptococcus sp. M334
Streptococcus sp. oral taxon 056 str.
Streptococcus sp. oral taxon 058 str.
Streptococcus sp. oral taxon 071 str.
Streptococcus thermophilus (strain
Streptococcus urinalis 2285-97
Streptococcus vestibularis ATCC 49124
Tannerella forsythia (strain ATCC
Bacteroides
forsythus
Tepidanaerobacter acetatoxydans (strain
Thermincola potens (strain JR)
Thermovirga lienii (strain ATCC BAA-
Treponema sp. JC4
Turicibacter sanguinis PC909
Turicibacter sp. HGF1
Victivallis vadensis ATCC BAA-548
Weeksella virosa (strain ATCC
Weissella confusa LBAE C39-2
Weissella paramesenteroides ATCC
Weissella thailandensis fsh4-2
Arabidopsis lyrata subsp. lyrata (Lyre-
Arabidopsis lyrata subsp. lyrata (Lyre-
Arabidopsis thaliana (mouse-ear cress)
Arabidopsis thaliana (mouse-ear cress)
Arabidopsis thaliana (mouse-ear cress)
Arachis hypogaea (peanut)
Arachis hypogaea (peanut)
Arachis hypogaea (peanut)
Arachis hypogaea (peanut)
Brachypodium sylvaticum (false brome)
Brassica campestris (field mustard)
Brassica juncea (Indian mustard)
Camellia oleifera
Camellia oleifera
Camellia oleifera
Camellia oleifera
Camellia oleifera
Capsicum annuum (bell pepper)
Capsicum chinense (Scotch bonnet)
Capsicum frutescens (cayenne papper)
Chimonanthus praecox
Chlamydomonas reinhardtii
Citrus sinensis (sweet orange) (Citrus
aurantium var. sinensis)
Coccomyxa subellipsoidea C-169
Cocos nucifera (coconut)
Cocos nucifera (coconut)
Cocous nucifera (coconut)
Cuphea calophylla subsp. mesostemon
Cuphea calophylla subsp. mesostemon
Cuphea hookeriana (cigar plant)
Cuphea hookeriana (cigar plant)
Cuphea hookeriana (cigar plant)
Cuphea hookeriana (cigar plant)
Cuphea lanceolata (cigar flower)
Cuphea lanceolata (cigar flower)
Cuphea lanceolata (cigar flower)
Cuphea lanceolata (cigar flower)
Cuphea palustris
Cuphea palustris
Cuphea viscosissima
Cuphea viscosissima
Cuphea viscosissima
Cuphea wrightii (Wright's waxweed)
Cuphea wrightii (Wright's waxweed)
Garcinia mangostana
Garcinia mangostana
Garcinia mangostana
Glycine max (soybean) (Glycine hispida)
Glycine max (soybean) (Glycine hispida)
Glycine max (soybean) (Glycine hispida)
Haematococcus pluvialis
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Helianthus annuus (common sunflower)
Iris germanica (flag) (fleur-de-lis)
Iris germanica (flag) (fleur-de-lis)
Iris germanica (flag) (fleur-de-lis)
Iris tectorum
Iris tectorum
Iris tectorum
Jatropha curcas
Macadamia tetraphylla
Macadamia tetraphylla
Medicago truncatula (barrel medic)
Myristica fragrans (nutmeg)
Myristica fragrans (nutmeg)
Nicotiana tabacum (common tobacco)
Perilla frutescens (beefsteak mint)
Populus tomentosa (Chinese white
Triticum aestivum (wheat)
Ulmus americana (American elm)
Umbellularia californica (California bay
A method of altering the specificity of a plant acyl-ACP TE for at least one of its substrates is also provided. For example, the specificity of a plant acyl-ACP TE for at least one of its substrates can be increased or decreased, even eliminated. The method comprises introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167, such as from about amino acid 118 to about amino acid 167, and/or a substrate specificity-altering mutation in the region corresponding to amino acids 73-85, such as from about amino acid 73 to about amino acid 85, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2; see, SEQ ID NO:3 in
In view of the foregoing, a method of altering the level of activity of a plant acyl-ACP TE is also provided. For example, the activity level, e.g., thioesterase activity level, such as the total amount of fatty acids produced, of the plant acyl-ACP TE can be increased or decreased compared to the activity level of the corresponding wild-type TE. An alteration in the level of activity can be an increase in fatty acid production or a decrease in fatty acid production, irrespective of whether or not the mol percentage of each fatty acid changes or not. Preferably, even desirably, the level of activity of the plant acyl-ACP TE is increased, rather than decreased. The method comprises (i) mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Mutating amino acid 173 to F, mutating amino acid 176 to L, mutating amino acid 205 to F, or a combination of two or more of the foregoing can alter the level of activity of the plant acyl-ACP TE, such as increase the level of activity of the plant acyl-ACP TE. The method can comprise mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Mutating amino acid 133 to F or L can increase production of C8 fatty acids, mutating amino acid 133 to V or A can increase production of C14/16 fatty acids, mutating amino acid 139 to I can increase production of C8-C12 fatty acids (e.g., C8 fatty acids), mutating amino acid 139 to N can increase production of C14/16 fatty acids, mutating amino acid 142 to A and mutating amino acid 143 to S can increase production of C8 fatty acids, and mutating both of amino acids 142 and 143 to R can increase production of C14/16 fatty acids. The method can further comprise mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Mutating amino acid 110 to F can increase production of C4/6 fatty acids, mutating amino acid 110 to L can increase production of C8 fatty acids, mutating amino acid 110 to V can increase production of C14/16 fatty acids, mutating amino acid 184 to F or L can increase production of C8-C12 fatty acids (e.g., C8 fatty acids), and mutating amino acid 184 to I can increase production of C14/16 fatty acids.
Also in view of the foregoing, an isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE derived from a wild-type acyl-ACP TE, such as an acyl-ACP TE from
Further in view of the foregoing, an isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a chimeric Cuphea viscosissima acyl-ACP TE gene, which comprises a segment of another acyl-ACP TE gene, is provided. Any suitable acyl-ACP TE gene can serve as the source of the segment that is used to replace the segment of the wild-type Cuphea viscosissima acyl-ACP TE gene (see, e.g.,
Still further in view of the foregoing, an isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167, such as from about amino acid 118 to about amino acid 167, and/or amino acids 73-85, such as from about amino acid 73 to about amino acid 85, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2), is provided. Alternatively, the region can correspond to amino acids 110-184, such as from about amino acid 110 to about amino acid 184, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Also, alternatively, the region can correspond to amino acids 110-205, such as from about amino acid 110 to about amino acid 205, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Any suitable plant acyl-ACP TE gene can be mutated (see, e.g.,
Still yet another isolated or purified nucleic acid molecule is provided. The isolated or purified nucleic acid molecule comprises a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises (i) a level of activity-altering mutation (e.g., a mutation that alters the total amount of fatty acids produced, such as increases the total amount of fatty acids produced) of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Any suitable plant acyl-ACP TE gene can be mutated (see, e.g.,
Mutations, such as substitutions, insertions, deletions, and/or side chain modifications, can be introduced into the nucleotide and amino acid sequences of the acyl-ACP TE using any suitable technique known in the art, including site-directed mutagenesis (Wu, ed., Meth. Enzymol. 217, Academic Press (1993)). Alternatively, domains can be swapped between acyl-ACP TE genes (for example, when creating chimeras). Non-naturally occurring nucleotides and amino acids also can be used. Mutations to the nucleotide sequence should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structures. The mutant or chimeric acyl-ACP TE may have altered substrate specificity, e.g., reacts with an acyl-ACP substrate that differs in chain length, degree of saturation, or presence/absence of a side group (e.g., methyl group), from that which is acted upon by the wild-type (also referred to as “native”) acyl-ACP TE. Alternatively, the mutant or chimeric acyl-ACP TE may have altered relative substrate specificity between two or more substrates, both of which are acted upon by the wild-type acyl-ACP TE. Both types of alterations in substrate specificity are encompassed by references to alterations of substrate specificity and substrate specificity-altering mutations herein. Alternatively or additionally to altered substrate specificity, the mutant or chimeric acyl-ACP TE may have an altered activity level, e.g., level of thioesterase activity, such as the total amount of fatty acids produced, including increased or decreased activity. Altered substrate specificity and altered activity can be detected by expression of the mutant thioesterase in E. coli, for example, and assay of enzyme activity.
A nucleotide sequence encoding all or a part of an acyl-ACP TE can be chemically synthesized, such as by the phosphoramidite method (Beaucage et al., Tetrahedron Letters 22: 1859-1869 (1981); and Matthes et al., EMBO J. 3: 801-805 (1984)). Polynucleotides can be synthesized, purified, annealed to their complementary strand, ligated, and then, optionally, cloned into suitable vectors.
The isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant/chimeric acyl-ACP TE can be a vector. The vector can contain, and preferably does contain, transcription and translation control regions. A promoter can be constitutive or regulatable, such as inducible. Additional sequences that can be present in the vector include pre-processing sequences, such as transit peptide sequences and plastid transit peptide sequences.
The acyl-ACP TEs and mutant/chimeric acyl-ACP TEs identified herein can be used in whole or in part as probes in hybridization assays to identify other TEs that can be used in the methods described herein. The TEs or fragments thereof also can be used as primers to amplify target DNA, such as by polymerase chain reaction (PCR) and other nucleic acid amplification methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., eds., Short Protocols in Molecular Biology, 5th ed., John Wiley & Sons (2002).
The nucleic acid molecule comprising a nucleotide sequence encoding an acyl-ACP TE or a mutant/chimeric acyl-ACP TE can be introduced into a host cell or a host organism using any suitable technique as is known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); Ausubel et al., eds., Short Protocols in Molecular Biology, 5th ed., John Wiley & Sons (2002). Such methods include microinjection, DNA particle bombardment, electroporation, liposome fusion, Agrobacterium-mediated transformation, and methods exemplified herein. Depending on the host cell or the host organism, one method can be preferred over another as readily appreciated by one of ordinary skill in the art. The nucleotide sequence can be codon-optimized for the recipient host cell or organism.
In view of the above, a host cell comprising an above-described isolated or purified nucleic acid molecule is also provided. The host cell or organism can be any suitable host cell or organism. The host cell or organism can be prokaryotic or eukaryotic, unicellular or multicellular, and undifferentiated or differentiated. If large-scale production of short-chain fatty acids is desired, e.g., as a source of biofuel, bacteria (see, e.g., U.S. Pat. App. Pub. No. 2012/0164700, which discloses examples of cyanobacteria, and U.S. Pat. App. Pub. No. 2009/0298143, which discloses methods of expression in bacteria, and both of which are hereby incorporated by reference for their teachings regarding same), yeast (see, e.g., U.S. Pat. App. Pub. No. 2011/0294174, which discloses examples of yeast in Table 26 and other fungi in Table 27 and which is hereby incorporated by reference for its teachings regarding same), and algae (see, e.g., U.S. Pat. App. Pub. No. 2011/0294174, which discloses examples of algae in Table 1 and which is hereby incorporated by reference for its teachings regarding same; also, see U.S. Pat. No. 7,935,515 and U.S. Pat. App. Pub. No. 2012/0164700, which disclose methods of expressing TEs in microalgae and examples of microalgae and which are hereby incorporated by reference for their teachings regarding same; see, also, U.S. Pat. App. Pub. No. 2009/0317878, which is hereby incorporated by reference for its teachings regarding expression of genes in algae) can be preferred. A preferred bacterium is Escherichia coli, in particular the strain K27. A preferred yeast is Saccharomyces cerevisiae. Alternatively, a crop plant (e.g., maize), such as an oilseed crop plant or a seed cell thereof, can be preferred (see, e.g., U.S. Pat. No. 7,504,563, which discloses expression of a nucleic acid encoding a thioesterase in soybean seed and which is incorporated herein for its teachings regarding same). See, also, U.S. Pat. App. Pub. No. 2010/0154293, which discloses other examples of host cells in paragraph [0080] and which is incorporated herein by reference for its teachings regarding same.
Fatty acids can be harvested, or otherwise collected (e.g., isolation from media containing bacteria that secrete the fatty acids), from host cells or organisms by any convenient method. Cells can be lysed/disrupted (e.g., heat, enzymes, ultrasound, mechanical lysis, osmotic shock, acid/base addition, or infection with a lytic virus), and fatty acids can be separated from cell mass by centrifugation and extraction (e.g., extraction with hydrophobic solvent, liquefaction, supercritical CO2 extraction, or hexane extraction after freeze-drying and pulverization) and further processed/refined as necessary. See, e.g., U.S. Pat. No. 7,935,515 and U.S. Pat. App. Pub. No. 2012/0135479, which are incorporated specifically by reference for their teachings regarding same.
An isolated or purified mutant acyl-ACP TE derived from a wild-type acyl-ACP TE, such as an acyl-ACP TE from
An isolated or purified chimeric Cuphea viscosissima acyl-ACP TE, which comprises a segment of another acyl-ACP TE, is also provided. Any suitable acyl-ACP TE can serve as the source of the segment that is used to replace the segment of the wild-type Cuphea viscosissima acyl-ACP TE (see, e.g., an acyl-ACP TE from
An isolated or purified mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167, such as from about amino acid 110 to about amino acid 167, and/or amino acids 73-85, such as from about amino acid 73 to about amino acid 85, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2), is also provided. Alternatively, the region can correspond to amino acids 110-184, such as from about amino acid 110 to about amino acid 184, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Also, alternatively, the region can correspond to amino acids 110-205, such as from about amino acid 110 to about amino acid 205, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Yet another isolated or purified mutant plant acyl-ACP TE is provided. The isolated or purified mutant plant acyl-ACP TE comprises (i) a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2) and (ii) a substrate specificity-altering mutation in the region corresponding to amino acids 118-167, such as from about amino acid 118 to about amino acid 167, and/or amino acids 73-85, such as from about amino acid 73 to about amino acid 85, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Alternatively, the region can correspond to amino acids 110-184, such as from about amino acid 110 to about amino acid 184, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). Also, alternatively, the region can correspond to amino acids 110-205, such as from about amino acid 110 to about amino acid 205, of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can comprise a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2). The mutant TE can further comprise a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2).
Once sequenced, polypeptides can be synthesized using methods known in the art, such as, for example, exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, and classical solution synthesis. See, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149 (1963), and Stewart and Young in Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). Automated peptide synthesizers are commercially available, as are services that make peptides to order.
The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.
This example describes the functional characterization of diverse acyl-ACP TEs rationally chosen based on phylogenetic classification of the TEs.
Sequences from Family TE14 (Cantu et al. (2010), supra) in the ThYme database (on the worldwide web at enzyme.cbirciastate.edu) were downloaded from the GenBank (Benson et al., Nucleic Acids Res. 39 (suppl. 1): D32-D37 (2011)) and UniProt (UniProt Consortium: The universal protein resource (UniProt) in 2010, Nucleic Acids Res. 38: D142-D148 (2010)) databases. Fragments and incomplete sequences were removed, yielding 360 acyl-ACP TE sequences. A multiple sequence alignment (MSA) was generated from catalytic domains of these sequences using MUSCLE 3.6 (Edgar, Nucleic Acids Res. 32: 1792-1797 (2004)) with default parameters. An unrooted phylogenetic tree based on the MSA was built using Molecular Evolutionary Genetics Analysis 4 (MEGA4) (Tamura et al., Mol. Biol. Evol. 24: 1596-1599 (2007)).
The minimum evolution algorithm was used due to its high effectiveness with large data sets (Desper et al., J. Comput. Biol. 9: 687-705 (2002)), gaps were subjected to pairwise deletion, and an amino acid Jones-Taylor-Thornton (JTT) (Jones et al., Comput. Appl. Biosci. 8: 275-282 (1992)) distance model was chosen. The phylogenetic tree was further verified by a bootstrap test with 1,000 replicates. The bootstrapped consensus tree was qualitatively analyzed and broken into apparent subfamilies. Statistical analysis was conducted to show that all sequences within a subfamily were more closely related to each other than to sequences in other subfamilies. Based on the MSA, JTT distances between all sequences were calculated and arranged into a j×j matrix, where j is the total number of sequences. Inter-subfamily distances and variances were determined using this matrix. For each apparent subfamily, a smaller k×k matrix, where k is the number of sequences in a given subfamily, was calculated. From this, intra-subfamily mean distances and variances were determined. These values were applied to the following equation to determine z:
where
A z-value >3.3 between two subfamilies shows that the difference between them is statistically significant to p<0.001. If a z-value between two apparent subfamilies were <3.3, alternative apparent subfamilies were chosen and/or individual sequences were removed, and the statistical calculations were repeated. Subfamilies were finally defined with a phylogenetic tree in which all z-values exceeded 3.3, sometimes leaving some sequences outside any subfamily (i.e. non-grouped sequences) (see Table 2).
Cuphea palustris
Ulmus
Americana
Iris germanica
Iris germanica
Sorghum bicolor
Sorghum bicolor
Cocos numfera
Cocos numfera
Cocos numfera
Cuphea
viscosissima
Cuphea
viscosissima
Cuphea
viscosissima
Elaeis
guineensis
Physcomitrella
patens
Sorghum bicolor
Sorghum bicolor
Micromonas
pusilla
Desulfovibrio
vulgaris
Bacteroides
fragilis
Parabacteroides
distasonis
Bacteroides
thetaiotaomicron
Clostridium
perfringens
Clostridium
asparagiforme
Bryantella
formatexigens
Geobacillus sp.
Streptococcus
dysgalactiae
Lactobacillus
brevis
Lactobacillus
plantarum
Anaerococcus
tetradius
Bdellovibrio
bacteriovorus
Clostridium
thermocellum
aA: Functionally characterized TEs; B: TE does not group near characterized TEs and/or no organism lipid profile information is available; C: TEs cloned from organisms known to produce MCFAs; D: Organism's lipid profile used and predominant fatty acid constituents identified in the organism are listed in parentheses.
bThe data are represented as mean ± standard error (n = 4).
cAll but the three C. nucifera sequences were codon-optimized for expression in E. coli.
dTransit peptides were removed from all plant sequences.
eAcyl-ACP TEs with known crystal structures.
All sequences within individual subfamilies were aligned using MUSCLE 3.6, and rooted phylogenetic trees were built in MEGA4 with the same tree and bootstrap parameters as described above. A few sequences from another subfamily (that with the highest z-value) were chosen to root individual subfamily trees.
A total of 360 amino acid sequences belonging to Family TE14 (Cantu et al. (2010), supra) were subjected to phylogenetic analysis and grouped into subfamilies. A subfamily is defined as having at least five sequences from different species, and it must pass the statistical tests described in the experimental procedures. Ten subfamilies met these criteria, accounting for 326 TE sequences; in addition 34 TE sequences could not be grouped into any of these subfamilies. All z-values were >3.4, ranging from 3.41 to 29.7, and mean distances between different subfamilies were larger than those within subfamilies.
Family TE14 contains acyl-ACP TEs that had previously been characterized from plants and classified into two types, FatA and FatB (Jones et al. (1995), supra). Of the ten subfamilies identified, Subfamilies A, B, and C are comprised of acyl-ACP TEs found in plants. All experimentally characterized sequences previously classified as FatB acyl-ACP TEs make up ˜25% of Subfamily A, which contains 81 angiosperm-sourced sequences. The coconut and C. viscosissima acyl-ACP TEs identified also belong to this subfamily. Subfamily B, which comprises 21 sequences primarily sourced from angiosperms as well as from the moss Physcomitrella patens, represents a potentially novel plant acyl-ACP TE subfamily with no previous experimental or phylogenetic characterization. Plant FatA acyl-ACP TEs, which act on long-chain acyl-ACP molecules, especially oleoyl-ACP (Jones et al. (1995), supra), belong to the 32-member Subfamily C. As with Subfamily B, the six green algal sequences from Chlamydomonas, Ostreococcus, and Micromonas that comprise Subfamily D have not been experimentally characterized.
Unlike several plant acyl-ACP TEs, no bacterial acyl-ACP TEs had been previously functionally characterized. A total of 186 bacterial acyl-ACP TE sequences were classified into six subfamilies (Subfamily E-Subfamily J). All 17 acyl-ACP TE sequences from gram-negative bacteria are in Subfamily E, which includes sequences from halophilic (Salinibacter and Rhodothermus), sulfate-reducing (Desulfovibrio, Desulfohalobium, and Desulfonatronospira), chemo-organotrophic (Spirosoma), metal-reducing (Anaeromyxobacter, Geobacter, and Pelobacter), and marine (Microscilla) bacteria. Subfamily F consists of 24 sequences, mainly from Bacteroides but also from other related bacteria. Protein Data Bank (PDB) structure 2ESS, obtained from a structural genomic effort, is part of this subfamily. Subfamily G and Subfamily H have 31 and 27 sequences, respectively, primarily from Clostridium. Subfamily I is comprised of eight sequences from six genera. Gram-positive lactic acid bacteria, almost completely from the genera Lactobacillus, Enterococcus, and Streptococcus, are part of Subfamily J (79 sequences). PDB:2OWN, the second bacterial acyl-ACP TE structure obtained from a structural genomic effort, appears in this family. Although the two known Family TE14 crystal structures (PDB:2ESS in Subfamily F and PDB:2OWN in Subfamily J) are from organisms in widely separated subfamilies, they are highly similar, as may be expected since they are members of the same enzyme family.
Some Family TE14 sequences are not grouped into any subfamily because their inclusion decreased z-values below acceptable limits. These include two plant and four moss sequences adjacent to Subfamilies A and C, and 28 bacterial sequences more closely related to Subfamilies E to I. No experimental work had previously been done on any of these sequences.
Upon generating the phylogenetic relationships among the 360 acyl-ACP TE sequences predicted or experimentally placed in Family TE14, 25 were chosen for experimental characterization. Of these, the cDNA for 24 was synthesized, while the cDNA of the Elaeis guineensis (oil palm) acyl-ACP TE was isolated from a phage cDNA library previously constructed from mRNA isolated from the developing fruit of Indonesian-sourced oil palm.
The selection of acyl-ACP TEs to characterize was based upon the primary structure-based phylogenetic relationships among the enzymes, along with knowledge of the fatty acid profile of the source organisms of these acyl-ACP TEs. Briefly, at least one TE was characterized from each of the ten subfamilies except for Subfamily C, whose members appear to be specific for oleoyl-ACP substrates. For subfamilies that contain acyl-ACP TEs originating from organisms without any known fatty acid data, or from organisms where acyl-ACP TEs were not previously characterized, acyl-ACP TE sequences that are evolutionarily distant from each other within each subfamily were selected for further investigation. For example, within Subfamily A there are two distinct and separate groupings of acyl-ACP TEs that are derived from the Poaceae family, for which there is no functional characterization (see Table 2). One grouping contains one sorghum acyl-ACP TE sequence (GenBank:EER87824) and the other contains two (GenBank:EER88593 and GenBank:EES04698). To explore this structural divergence as an indicator of potential functional divergence in substrate specificities, one each of these Subfamily A sorghum acyl-ACP TEs (GenBank:EER87824 and GenBank:EER88593) and the two Subfamily B sorghum acyl-ACP TEs were expressed and functionally characterized.
This example describes the cloning of acyl-ACP TEs from Cocos nucifera (coconut) and Cuphea viscosissima.
Coconut fruits of different developmental stages were obtained from the USDA-ARS-SHRS National Germplasm Repository (Miami, Fla., USA). Seeds of C. viscosissima were obtained from the North Central Regional Plant Introduction Station (NCRPIS, Ames, Iowa, USA). They were treated overnight with 0.1 mM gibberellic acid and then germinated in a growth chamber (Environmental Growth Chambers, Chagrin Falls, Ohio) with 12 hours of illumination at 25° C. followed by 12 hours of darkness at 15° C. Seedlings were transplanted into soil and cultivated at NCRPIS. Seeds at different developmental stages were collected and flash-frozen in liquid nitrogen.
Acyl-ACP TE cDNAs were cloned from C. viscosissima and coconut via a homologous cloning strategy. MSAs of plant TE14 sequences revealed two conserved regions (RYPTWGD [SEQ ID NO: 7] and NQHVNNVK [SEQ ID NO: 8]), from which two degenerate primers, DP-F3 (5′-AGNTAYCCNACNTGGGGNGA-3′ [SEQ ID NO: 9]) and DP-R3 (5′-TACTTNACRTTRTTNACRTGYTGRTT-3′ [SEQ ID NO: 10]), were designed. RNA was extracted from endosperm of nearly mature coconuts and immature seeds of C. viscosissima using the total RNA (plant) kit (IBI Scientific, Peosta, Iowa, USA). RNA was reverse-transcribed to cDNA using the SuperScript™ first-strand synthesis system for RT-PCR kit (Invitrogen, Carlsberg, Calif., USA). PCR was performed in a 50-μL reaction mixture containing 20 ng cDNA, 1×Pfx buffer, 1 mM MgSO4, 0.3 mM dNTP, 5.12 μM DP-F3 and DP-R3 primers, and 0.5 U Pfx polymerase (Invitrogen) using a cycling program of 94° C. for four minutes, 35 cycles of 94° C. for 30 seconds, 52° C. for 30 seconds and 72° C. for 45 seconds, and a final extension step of 72° C. for five minutes. The expected ˜350-bp products were identified by agarose gel electrophoresis, and their DNA bands were recovered using the QiaQuick gel extraction kit (Qiagen, Valencia, Calif., USA) and cloned into the pENTR TOPO TA vector (Invitrogen). Using primers designed from the sequences of the cloned 350-bp fragments, the 5′- and 3′-ends of the cDNAs were obtained using the SMARTer RACE (rapid amplification of the cDNA ends) cDNA amplification kit (Takara Bio, Otsu, Japan).
For each acyl-ACP TE sequence, the full-length cDNA, minus the N-terminal chloroplast transit peptide, was amplified by PCR with primers engineered to introduce Bam HI and Eco RI restriction sites at the 5′- and 3′-ends, respectively. The PCR-amplified products were digested with Bam HI and Eco RI and cloned into the corresponding restriction sites of the pUC57 vector, which placed the acyl-ACP TE sequence under the transcriptional control of the lacZ promoter. The sequence of each construct was confirmed by sequencing both strands. Confirmed expression vectors of coconut genes were transformed into E. coli strain K27, while sequences of C. viscosissima acyl-ACP TEs were synthesized after being codon-optimized for expression in E. coli using the OptimumGene codon optimization program provided by GenScript USA (Piscataway, N.J., USA).
MCFAs are abundant in the oil produced in fruits of coconut (i.e., predominantly C12 and C14 and a small amount (0.2-1%) of C6 fatty acids (Kumar et al., J. Food Qual. 32: 158-176 (2009); Kumar et al., Indian Coconut J. 37: 4-14 (2006); and Kumar et al., Trop. Agr. 81: 34-40 (2004)) and seeds of C. viscosissima (i.e., predominantly C8 and C10 fatty acids (Phippen et al., Ind. Crop Prod. 24: 52-59 (2006)). Therefore, acyl-ACP TEs in the seeds of these species are predicted to be specific for medium-chain acyl-ACPs. Acyl-ACP TE sequences were isolated from coconut and C. viscosissima by a homologous cloning strategy. Using degenerate primers, which were designed from conserved regions of plant TE14 family enzymes, a 350-bp fragment in the middle of the mRNAs was amplified from cDNA generated from both developing coconut endosperm and C. viscosissima seeds. Sequencing of cloned PCR products identified three new acyl-ACP TE sequences each from coconut and C. viscosissima. The full-length cDNA sequences were obtained by RACE for three acyl-ACP TEs [CnFatB1 (JF338903), CnFatB2 (JF338904), and CnFatB3 (JF338905)] from coconut and three [CvFatB1 (JF338906), CvFatB2 (JF338907), and CvFatB3 (JF338908)] from C. viscosissima.
The predicted open reading frames of coconut and C. viscosissima acyl-ACP TE cDNAs were identified. They encode pre-proteins of 412 to 423 amino acids, with calculated molecular weights of 45.8 to 46.5 kDa and theoretical pIs of 6.4 to 8.8. Plant acyl-ACP TEs are nuclear-encoded, plastid-targeted proteins with an N-terminal plastid-targeting peptide extension (Voelker et al. (1992), supra). For each of the cloned coconut and C. viscosissima acyl-ACP TEs, the putative plastid-targeting peptide cleavage site was located on the N-terminal side of the conserved sequence LPDW, as proposed for many other plant acyl-ACP TEs (Jones et al. (1995), supra; Sanchez-Garcia (2010), supra; Dormann et al. (1995), supra; Jha et al., Plant Physiol. Biochem. 44: 645-655 (2006); and Moreno-Perez et al., Plant Physiol. Biochem. 49: 82-87 (2011)). These yield predicted mature proteins of 323 to 331 amino acid residues (Huynh et al., Plant Physiol. Biochem. 40: 1-9 (2002)), with calculated molecular weights of 36.6 to 37.5 kDa and theoretical pIs of 5.4 to 7.3. Alignment of the deduced amino acid sequences of coconut and C. viscosissima acyl-ACP TE cDNAs showed that, except for the plastid-targeting peptide sequences and very near the C-terminus, the sequences are co-linear and share very high identity (63-86%) within a species. These sequences cluster within Subfamily A.
This example describes in vivo activity assays.
E. coli strain K27 contains a mutation in the fadD gene impairing β-oxidation of fatty acids, which results in the accumulation of free fatty acids in the growth medium (Klein et al., Eur. J. Biochem. 19: 442-450 (1971); and Overath et al., Eur. J. Biochem. 7: 559-574 (1969)). Each TE was expressed in E. coli K27, and free fatty acids that accumulated in the medium were extracted and analyzed. Four colonies for each construct were independently cultured in 2 mL LB medium supplemented with 100 mg/L carbicillin in 17-mL culture tubes. When the culture reached an OD600 of ˜0.7, the growth medium was replaced with 3 mL of M9 minimal medium (47.7 mM Na2HPO4, 22.1 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, and 0.1 mM CaCl2) supplemented with 0.4% glucose and 100 mg/l carbicillin, and 10 μM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce acyl-ACP TE expression. After 40 hours of cultivation, cells were pelleted, and free fatty acids in the supernatant were extracted essentially following a previously described method (Voelker et al., J. Bacteriol. 176: 7320-7327 (1994); and Mayer et al., BMC Plant Biol. 2007: 7 (2007)). Briefly, 2 mL of culture supernatant was supplemented with 10 μg heptanoic acid (7:0), 10 μg undecanoic acid (11:0), and 20 μg heptadecanoic acid (17:0) (Sigma-Aldrich, St. Louis, Mo., USA) as internal standards. The mixture was acidified with 20 μL of 1 M HCl, and 4 mL chloroform-methanol (1:1 vol/vol) was used to recover the fatty acids from the medium. After vortexing for 10 minutes and centrifuging at 1000×g for four minutes, the lower chloroform phase was transferred to a new tube and evaporated under a stream of N2 gas until the samples were concentrated to ˜300 μL. Samples (1 μL) were analyzed on an Agilent Technologies (Santa Clara, Calif., USA) 6890 Series gas chromatograph (GC) system used with an Agilent 5973 mass selective detector equipped with an Agilent CP-Wax 58 FFAP CB column (25 mm×0.15 mm×0.39 mm). The GC program followed an initial temperature of 70° C. for two minutes, ramped to 150° C. at 10° C./minute and held for three minutes, ramped to 260° C. at 10° C./minute, and held for 14 minutes. Final quantification analysis was performed with AMDIS software (National Institute of Standards and Technology). Determination of C4 to C8, C10 to C12, and >C12 fatty acid concentrations was based on the fatty acid internal standards 7:0, 11:0, and 17:0, respectively. The total concentration of fatty acids produced by each acyl-ACP TE was obtained by subtracting the concentration of fatty acid produced by E. coli expressing a control plasmid (pUC57) lacking a TE from that produced by E. coli expressing a given acyl-ACP TE sequence from the same vector. The three most abundant fatty acids produced by the control strain were 8:0 (2.0 nmol/ml), 14:0 (3.5 nmol/ml), and 16:0 (3.1 nmol/ml), and their levels were minimal compared to strains expressing acyl-ACP TEs. Compared to GC analyses of fatty acids after derivatization (e.g., methylation or butylation), the GC-MS method used non-derivatized free fatty acids, which is better optimized for analyzing short-chain fatty acids (e.g., 4:0, 6:0, 8:0, 10:0, 12:0, and 14:0). However, this method may be less sensitive for longer-chain fatty acids (e.g., 18:0 and 18:1).
Analysis of free fatty acids revealed possible peaks characteristic of 2-tridecanone. To further confirm this identification, retention times and MS spectra of the peaks in each sample were compared to a 2-tridecanone standard (Sigma-Aldrich).
All isolated acyl-ACP TE cDNAs were expressed in E. coli strain K27. Secreted fatty acids were analyzed with GC-MS, and the total fatty acid yield in the medium was used to represent the in vivo activities of these enzymes on acyl-ACPs, though it remains possible that some of these enzymes might also hydrolyze acyl-CoAs (Othman et al., Biochem. Soc. Trans. 28: 619-622 (2000)).
A total of 13 acyl-ACP TEs from Subfamily A were characterized, including single acyl-ACP TEs from Cuphea palustris (GenBank:AAC49179), U. americana (GenBank:AAB71731), and oil palm (E. guineensis, GenBank:AAD42220), two each from Iris germanica (GenBank:AAG43857 and GenBank:AAG43858) and Sorghum bicolor (GenBank:EER87824 and GenBank:EER88593), and three each from coconut and C. viscosissima. Total fatty acid concentrations produced by these acyl-ACP TEs are listed in Table 1, and the resulting fatty acid compositions are shown in
C. palustris acyl-ACP TE produced 97 mol % 8:0 and only 0.8 mol % 10:0 fatty acids (
The CvFatB1 and CvFatB3 TEs, for which corresponding cDNAs were isolated from the developing seeds of C. viscosissima produced MCFAs in E. coli, and CvFatB1 shows substrate specificity consistent with the fatty acid constituents present in the seed oil. The relative distributions of 8:0 and 10:0 fatty acids differ; CvFatB1 produced twice as much 8:0 compared to 10:0 fatty acid, whereas there is ˜fourfold more 10:0 fatty acid within C. viscosissima seed oil (Phippen et al., Ind. Crop Prod. 24: 52-59 (2006)).
Three acyl-ACP TEs from plant sources belonging to Subfamily B, including those from P. patens (GenBank:EDQ65090) and S. bicolor (GenBank:EER96252 and GenBank:EES11622), and one acyl-ACP TE from Subfamily D sourced from the alga Micromonas pusilla (GenBank:EEH52851), were similarly characterized. Total activity in E. coli expressing these acyl-ACP TEs varied from 9 to 380 nmol/mL (Table 1). These four acyl-ACP TEs showed similar substrate specificities, producing predominantly 14:0 (34-65 mol %) and 16:1 (23-37 mol %) fatty acids (
Eleven acyl-ACP TE sequences from Subfamilies E to J sourced from bacteria and three bacterial sequences that were not placed in any subfamily were characterized (Table 1 and
The accumulation of both unsaturated fatty acids and saturated fatty acids observed is consistent with the previous conclusion that the heterologously expressed acyl-ACP TEs can intercept both saturated and unsaturated intermediates of fatty acid biosynthesis of E. coli (Magnuson et al., Microbiol. Rev. 57: 522-542 (1993)). Many bacterial acyl-ACP TEs, such as those from Desulfovibrio vulgaris (GenBank:ACL08376, Subfamily E), L. brevis (GenBank:ABJ63754, Subfamily J), L. plantarum (GenBank:CAD63310, Subfamily J), and Bdellovibrio bacteriovorus (GenBank:CAE80300, no subfamily), are part of the pathway that produces noticeable amounts of the methylketone 2-tridecanone through enzymatic hydrolysis of 3-keto-tetradecanoyl-ACP followed by chemical decarboxylation. Interestingly, in the E. coli heterologous expression system used, six bacterial-sourced acyl-ACP TEs and three plant-sourced acyl-ACP TEs produced noticeable amounts (>1 nmol/mL) of methylketones, largely 2-tridecanone. The acyl-ACP TE from B. bacteriovorus (GenBank: CAE80300) produced the highest concentration of 2-tridecanone (9.4 nmol/mL, which was 3 mol % of the fatty acids produced, as shown in
Methylketones, such as 2-tridecanone, occur in the wild tomato species Solanum habrochaites subsp. Glabratum (Antonious, J. Environ. Sci. Health B 36: 835-848 (2001)), and their biosynthesis is catalyzed by two sequentially-acting methylketone synthases, MKS1 and MKS2. MKS2 is a thioesterase that catalyzes the hydrolysis of the 3-ketoacyl-ACP intermediate in fatty acid biosynthesis, and MKS1 catalyzes the decarboxylation of the released 3-keto acid to produce a methylketone (Ben-Israel et al., Plant Physiol. 151: 1952-1964 (2009); and Yu et al., Plant Physiol. 154: 67-77 (2010)). Heterologous expression of MKS2 in E. coli yields many methylketones, including 2-tridecanone (Yu et al. (2010), supra). However, MKS2 is not included in Family TE14; rather, it is included in Family TE9 (Cantu et al. (2010), supra). Although some Family TE14 members share very low, if any, significant sequence similarity (i.e., <15% identity) to MKS2, the data indicate that at least nine acyl-ACP TEs (e.g., B. bacteriovorus, GenBank:CAE80300) can catalyze the same reaction as MKS2 (i.e., hydrolysis of the thioester bond of 3-ketoacyl-ACP), and that the resulting product (3-keto acid) is further chemically or enzymatically decarboxylated to generate the methylketone.
This example describes statistical cluster analysis.
To classify acyl-ACP TEs based on their in vivo activities, the fatty acid composition data obtained from the in vivo expression of all TE sequences studied were used to perform statistical clustering analysis. The distance matrix was calculated using Euclidean distances, and Ward's method (Ward, J. Am. Stat. Assoc. 58: 236 (1963)) was used to perform agglomerative hierarchical clustering. The p-values were calculated via multiscale bootstrap re-sampling with 1,000 replicates (Suzuki et al., Bioinformatics 22: 1540-1542 (2006)).
All acyl-ACP TEs that were characterized were clustered into three classes: 1) Class I contains acyl-ACP TEs that mainly act on C14 and C16 substrates; 2) Class II has acyl-ACP TEs that have broad substrate specificities, with major activities toward C8 and C14 substrates; and 3) Class III comprises acyl-ACP TEs that predominantly act on C8 substrate (
Comparison between the specificity-based classification and the sequence-based phylogenetic tree indicates that the two classifications are not necessarily consistent with each other. Three phenomena were observed in this study. First, diverged sequences (variants in primary structure) from the same species do not necessarily differ in function. Second, similar sequences may have different substrate specificities. Third, sequences that belong to different subfamilies because they share low sequence identity can have very similar substrate specificities. Therefore, it is not reasonable to infer the substrate specificity of one acyl-ACP TE based on its sequence-based classification within the same subfamily. It is conceivable, therefore, that the change of substrate specificity is most likely caused by changes of only a few amino acid residues, and that many different combinations of residue changes could result in changed specificities (Jones (1995), supra). Bacterial orthologs provide access to additional functional diversity, both relative to acyl chain length specificity (e.g., shorter acyl chains, as short as four carbon atoms), as well as acyl chains that contain additional chemical functionalities (e.g., unsaturated acyl chains and acyl chains containing carbonyl groups).
This example describes the generation of random mutants and site-directed mutants of acyl-ACP TE from Bryantella formatexigens (EET61113).
DNA sequences for the wild-type acyl-ACP TE from Bryantella formatexigens (EET61113; nucleotide sequence is SEQ ID NO: 23; amino acid sequence is SEQ ID NO: 24) was synthesized and cloned into pUC57 vector as previously. The random mutants were generated by error-prone PCR using primers designed on pUC57 vector (pUC57F: 5′-CTGCAAGGCGATTAAGTTGGGTAAC-3′ [SEQ ID NO: 11]; pUC57R: 5′-CGGCTCGTATGTTGTGTGGAAT-3′ [SEQ ID NO: 12]). The PCR was conducted in 40 tubes of reaction mixture (15 μl), which contained 1×PCR buffer, 0.2 mM dATP and dGTP, 1 mM dCTP and dTTP, 7 mM MgCl2, 0.1 mM MnCl2, 0.5 μl of each primers, 1.5 ng plasmid containing the thioesterase gene, and 0.15 U Taq DNA polymerase (Invitrogen), using a cycling program of 94° C. for 4 minutes, 31 cycles of 94° C. for 30 seconds, 52° C. for 30 seconds, and 72° C. for 1 minute, and a final extension step of 72° C. for 5 minutes. The PCR products were pooled together, purified with the QiaQuick gel extraction kit (Qiagen, Valencia, Calif., USA), digested with Bam HI and Eco RI, and then cloned into the corresponding restriction sites of the pUC57 vector. The constructed vectors containing mutant genes were transformed into E. coli K27 by electroporation.
Site-directed mutations were also introduced into wild-type acyl-ACP TE EET61113. Specifically, a mutant (designated TE20-N169Y), which contains a single N→Y mutation at amino acid position 169, and another mutant (designated TE20-52221), which contains a single S→I mutation at amino acid position 222, were generated.
This example describes the initial screening of acyl-ACP TE mutants generated in Example 5.
Mutants were screened on Neutral Red-containing media, which was M9 minimal medium (47.7 mM Na2HPO4, 22.1 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, and 0.1 mM CaCl2) solidified by 15 g/L agar and supplemented with 0.4% glucose, 100 mg/L carbicillin, 1 mM IPTG, and 40 ppm Neutral Red. This screening method is based on pH change in the media. Mutants with higher acyl-ACP TE activity will produce more free fatty acids, which decrease the pH of the colonies and generate a more intense red color. Briefly, after electroporation, an appropriate amount of culture was spread on the Neutral Red plates so that each plate would contain 100-200 colonies. The plates were incubated at 30° C. for two days, and then at room temperature for another 3-5 days. Eventually, the colonies that are more intensely red were selected for further characterization with GC-MS.
This example describes the further characterization of those mutants that were identified in Example 6 as producing more fatty acids.
Colonies that were more intensely red on the Neutral Red plate were assumed to produce more fatty acids. The activity and composition of the mutants were characterized as described above with slight modification. Instead of 20 μl of 1 M HCl, 200 μl of 1 M HCl were used to acidify the cell culture supernatant, which allowed recovery of more butanoic acid from the sample. Thus, the activity of wild-type thioesterase EET61113 in this Example was higher than the production in the above Examples.
The in vivo activities of 139 mutants have been determined so far. Their total activities are shown in
This example describes the construction of chimeric TEs.
CvFatB1 and CvFatB2 share 72% identity in their amino acid sequences, but have very different substrate specificities: CvFatB1 mainly produces C8 and C10 fatty acids, while CvFatB2 produces C14 and C16 fatty acids. Chimeric TEs were constructed using these two sequences to locate the region(s) that determine the substrate specificity of acyl-ACP TEs. Previously, CvFatB1 and CvFatB2 genes were codon-optimized, synthesized, and cloned into the pUC57 vector. Using the primers listed in Table 3, six fragments (I, II, III, IV, V, and VI) for each TE gene were generated (see
The chimeric TEs were constructed by re-assembling the six fragments to recreate the full-length thioesterase gene sequence by PCR, using a combination of fragments from either CvFatB1 or CvFatB2. PCR was performed in a 50-μL reaction mixture containing 10 ng of each fragment, 1× Phusion buffer, 0.2 mM dNTP, 0.5 μM pUC57F and pUC57R primers, and 1 Unit of Phusion high-fidelity DNA polymerase (New England Biolabs) using a cycling program of 98° C. for 2 minutes, 32 cycles of 98° C. for 10 seconds, 50° C. for 15 seconds, and 72° C. for 20 seconds, and a final extension step of 72° C. for 5 minutes. The expected full-length gene products were identified by agarose gel electrophoresis, recovered from the gel using the QiaQuick gel extraction kit (Qiagen, Valencia, Calif.) and cloned into the pUC57 vector using the Bam HI and Eco RI restriction sites. The sequence of each construct was confirmed by sequencing using primers pUC57F and pUC57R.
This example describes the use of sequence alignments to identify residues that may affect substrate-specificity.
A total of 27 representative acyl-ACP TE sequences, including both plant and bacterial TEs that were previously functionally characterized, were aligned using Vector NTI software (Invitrogen) with the default parameters. While Cysteine 264 was previously proposed to be a catalytic residue by Mayer et al. (J. Biol. Chem. 280: 3621-3627 (2005)), Cys264 was not conserved among these 27 TE sequences. The adjacent glutamic acid (Glu263), however, was conserved and predicted to be a catalytic residue (see
This example describes the use of site-directed mutagenesis to verify the residues that may affect substrate specificity.
Point mutants were generated from CvFatB2 with the QuikChange II site-directed mutagenesis kit (Agilent Technologies) according to manufacturer's instructions in order to test whether the predicted residues, which were identified in Example 9, affected the substrate specificity of acyl-ACP TEs. The residues of CvFatB2 were mutated to the corresponding residues in CvFatB1 with the exception of residues valine 110 (V110) and isoleucine 184 (I184), which were mutated to the bulkier residue, phenylalanine. The point mutations were introduced sequentially for the mutants that harbored multiple amino acid changes. All mutants were confirmed by sequencing. The results are shown in
This example describes the production of fatty acids in vivo by TE variants.
TE variants were analyzed in accordance with the method of Example 3. The results are shown in
This example describes the generation and fatty acid production analysis of TE mutants.
Protein sequences of CvFatB1 and CvFatB2 from Cuphea viscosissima, CnFatB2 and CnFatB3 from Cocos nucifera, UaFatB1 from Ulmus americana, and CpFatB1 from Cuphea hookeriana were subjected to multiple sequence alignments with Clustal W2 (on the worldwide web at ebi.ac.uk/Tools/msa/clustalw2/) (see
The acyl-ACP TE mutant library was generated by assembling 30 oligo primers in two rounds of PCR. The first round of PCR was conducted in 50 μL of reaction mixture containing 0.15 μM of each primer, 1×Taq PCR buffer, 0.4 mM dNTP, 3 mM MgCl2, and 1 Unit of Taq DNA polymerase (New England Biolabs, USA) using a cycling program of 95° C. for three minutes, 55 cycles of 95° C. for 15 seconds, 50° C. for 20 seconds, and 68° C. for 40 seconds, and a final extension step of 68° C. for five minutes. The second round of PCR was performed in eight tubes of 50 μL of reaction mixture containing 1×Taq PCR buffer, 2 μL of first-round PCR product as template, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 μM of each of the first and last primers, and 1 Unit Taq DNA polymerase using a cycling program of 95° C. for three minutes, 28 cycles of 95° C. for 15 seconds, 60° C. for 20 seconds, and 68° C. for 40 seconds, and a final extension step of 68° C. for five minutes. The second-round PCR products were pooled, separated by electrophoresis on 1% agarose gel, purified with the QiaQuick gel extraction kit (Qiagen, Valencia, Calif., USA), digested with Bam HI and Eco RI, and cloned into the corresponding restriction sites of the pUCHisGm vector (
The initial screening of the TE variants was conducted on Neutral Red plates, which contained M9 minimal medium (47.7 mM Na2HPO4, 22.1 mM KH2PO4, 8.6 mM NaCl, 18.7 mM NH4Cl, 2 mM MgSO4, and 0.1 mM CaCl2) solidified by 15 g/L agar and supplemented with 0.4% glucose, 100 mg/L carbenicillin, 2.5 mg/L gentamicin, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and 100 ppm Neutral Red dye. Neutral Red is a pH indicator that changes color to red when the pH drops below 6.8. Variants with higher TE activity will produce more free fatty acids, which will decrease the pH of the colonies and generate a more intense red color. Briefly, after electroporation, an appropriate amount of the culture was spread on the Neutral Red plates so that each plate had 300-500 colonies. The plates were incubated at 30° C. for three days. Approximately 98% of the colonies displayed light red color. Only about 2% of the colonies displayed intense red color.
In order to determine whether fatty acid production is correlated with the color of the colonies, 133 dark red colonies and 77 light red colonies were randomly picked from the Neutral Red plates for further analysis with GC-MS. For each colony expressing a TE mutant, the free fatty acids were extracted and analyzed, and the total fatty acid production was calculated.
Of the 133 strains that expressed dark red colonies, 75% produced more than 600 μM of fatty acids, 50% produced more than 1,000 μM of fatty acids, and 25% produced more than 1,200 μM of fatty acids. Only 25% of the strains that expressed dark red colonies produced less than 600 μM of fatty acids. In contrast, most of the strains that expressed light red colonies produced very small amounts of fatty acids (<100 μM). The maximum fatty acid production of the light red colonies was 264 μM, which was much lower than the production of most dark red colonies. These results indicate that there is a strong correlation between the color of the colonies and the total fatty acid production, validating the Neutral Red screening protocol for identifying strains that produce high levels of fatty acids.
In order to identify TE variants from the variant library that produced more fatty acids, 480 colonies were selected from the Neutral Red plate based upon the red-color colony phenotype, and their fatty acid production was analyzed using GC-MS. CnFatB3, CvFatB1, CnFatB2, UaFatB1, CvFatB2, and CpFatB1 were analyzed using GC-MS as controls; their total fatty acid production was 103, 243, 270, 352, 484 and 932 μM, respectively. Fatty acid production and composition for each variant was determined in vitro according to the method of Jing et al. (BMC Biochemistry 12: 44 (2011)) with slight modification. Colonies were picked from Neutral Red plates, inoculated into 700 μL of LB medium supplemented with 100 mg/L carbenicillin, and cultured overnight at 30° C. and an agitation rate of 250 rpm. The next morning 100 μL of the overnight culture were used to inoculate 2 mL M9 medium supplemented with 0.4% glucose, 100 mg/L carbenicillin, and 0.1 mM IPTG in a 16-mL test tube. After culturing at 30° C. and an agitation rate of 250 rpm for 48 hours, 1.5 mL of culture were used for fatty acid extraction. Following the addition of 50 μg heptanoic acid (7:0), 50 μg undecanoic acid (11:0), and 100 μg heptadecanoic acid (17:0) (Sigma-Aldrich, St. Louis, Mo., USA) as internal standards, the mixture was acidified with 500 μL of 1 M HCl, and 4 mL of chloroform-methanol (1:1 vol/vol) were used to recover the fatty acids from the culture. After vortexing for 10 minutes and centrifuging at 3,000× g for four minutes, the lower chloroform phase was transferred onto an anhydrous MgSO4 column to remove trace amounts of water and then evaporated under a stream of N2 gas until the samples were concentrated to ˜200 μL. These samples were subjected to GC-MS analysis. The fatty acid production of each acyl-ACP TE mutant was determined by subtracting the fatty acids produced by E. coli expressing a control plasmid (pUCHisGm) without any TE gene.
Among the 480 colonies analyzed, 156 colonies produced more than 1,000 μM total fatty acids. The highest fatty acid production observed was 1,695 μM (about 80% higher than that of CpFatB1). On a fatty acid weight basis, the highest productivity was 349 mg/L (about 2.6 fold higher than that of CpFatB1). The results are shown in
This example describes the sequence analysis of the TE mutants generated in Example 12.
A total of 192 acyl-ACP TE variants that produced fatty acids between 500 μM and 1,700 μM were selected for high-throughput sequencing; 177 of those were successfully sequenced. Among those that were sequenced, 147 variants had the identical sequence, referred to herein as TEGm162 (see
The fatty acid profiles of the 26 distinct variants and the six parental TEs were evaluated (see
Thus, in view of the above, the present invention provides the following:
A. A method of increasing production of fatty acids in a host cell or organism, which method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding an acyl-acyl carrier protein (ACP) thioesterase (TE) from Bryantella formatexigens, whereupon the production of fatty acids in the host cell or organism is increased.
B. The method of A, wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant.
C. A method of increasing production of short-chain fatty acids in a host cell or organism, which method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE derived from wild-type Bryantella formatexigens acyl-ACP TE, whereupon the production of short-chain fatty acids in the host cell or organism is increased and wherein the mutant acyl-ACP TE produces more short-chain fatty acids in the host cell or organism than the corresponding wild-type acyl-ACP TE.
D. The method of C, wherein the mutant acyl-ACP TE differs from wild-type Bryantella formatexigens acyl-ACP TE by two or more amino acid mutations comprising N169Y and S2221 and wherein the mutant acyl-ACP TE has increased thioesterase activity compared to wild-type Bryantella formatexigens acyl-ACP TE.
E. The method of C, wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant.
F. The method of D, wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant.
G. A method of making a mutant Bryantella formatexigens acyl-ACP TE, which method comprises making a mutant Bryantella formatexigens acyl-ACP TE comprising two or more amino acid mutations comprising N169Y and S222I, whereupon a mutant Bryantella formatexigens acyl-ACP TE is made.
H. The method of G, wherein the mutant Bryantella formatexigens acyl-ACP TE has increased thioesterase activity compared to a corresponding wild-type Bryantella formatexigens acyl-ACP TE.
I. An isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE, which is derived from wild-type Bryantella formatexigens acyl-ACP TE, comprises two or more amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type Bryantella formatexigens acyl-ACP TE, wherein the isolated or purified nucleic acid molecule can be a vector.
J. A host cell or organism comprising the isolated or purified nucleic acid molecule of I.
K. The host cell or organism of J, wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant.
L. An isolated or purified mutant acyl-ACP TE, which is derived from wild-type Bryantella formatexigens acyl-ACP TE, comprises two or more amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type Bryantella formatexigens acyl-ACP TE.
M. A method of making a chimeric Cuphea viscosissima acyl-ACP TE, which method comprises replacing a segment of a wild-type Cuphea viscosissima acyl-ACP TE with a segment of another acyl-ACP TE, whereupon a chimeric Cuphea viscosissima acyl-ACP TE is made.
N. The method of M, wherein the segment of another acyl-ACP TE gene is a segment of another Cuphea viscosissima acyl-ACP TE.
O. The method of M, which method comprises replacing a segment of a wild-type Cuphea viscosissima FatB1 (CvFatB1) gene with a segment of another acyl-ACP TE gene to produce a chimeric CvFatB1 gene or replacing a segment of a wild-type Cuphea viscosissima FatB2 (CvFatB2) gene with a segment of another acyl-ACP TE gene to produce a chimeric CvFatB2 gene.
P. The method of O, which method comprises replacing a segment of a wild-type CvFatB1 gene with a segment of a CvFatB2 gene to produce a chimeric CvFatB1 gene or replacing a segment of a wild-type CvFatB2 gene with a segment of a CvFatB1 gene to produce a chimeric CvFatB2 gene.
Q. The method of M, wherein the chimeric Cuphea viscosissima acyl-ACP TE (i) has a substrate specificity that differs from the corresponding wild-type Cuphea viscosissima acyl-ACP TE, (ii) produces a total amount of fatty acids that differs from the total amount of fatty acids produced by the corresponding wild-type Cuphea viscosissima acyl-ACP TE, or (iii) has a substrate specificity and produces a level of a fatty acid, both of which differ from the corresponding wild-type Cuphea viscosissima acyl-ACP TE.
R. An isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a chimeric Cuphea viscosissima acyl-ACP TE gene, which comprises a segment of another acyl-ACP TE gene, wherein the isolated or purified nucleic acid molecule can be a vector.
S. The isolated or purified nucleic acid molecule of R, wherein the segment of another acyl-ACP TE gene is a segment of another Cuphea viscosissima acyl-ACP TE gene.
T. The isolated or purified nucleic acid molecule of R, wherein the chimeric Cuphea viscosissima acyl-ACP TE gene is a chimeric FatB1 gene or a chimeric FatB2 gene.
U. The isolated or purified nucleic acid molecule of T, wherein the chimeric Cuphea viscosissima acyl-ACP TE gene is a chimeric FatB1 gene comprising a segment of a Cuphea viscosissima FatB2 gene or the chimeric Cuphea viscosissima acyl-ACP TE gene is a chimeric FatB2 gene comprising a segment of a Cuphea viscosissima FatB1 gene.
V. A host cell or organism comprising the isolated or purified nucleic acid molecule of R-U.
W. An isolated or purified chimeric Cuphea viscosissima acyl-ACP TE, which comprises a segment of another acyl-ACP TE.
X. The isolated or purified chimeric Cuphea viscosissima acyl-ACP TE of W, wherein the segment of another acyl-ACP TE is a segment of another Cuphea viscosissima acyl-ACP TE.
Y. The isolated or purified chimeric Cuphea viscosissima acyl-ACP TE of W, which is a chimera of the TE encoded by a FatB1 gene or a chimera of the TE encoded by a FatB2 gene.
Z. The isolated or purified chimeric Cuphea viscosissima acyl-ACP TE of Y, which is a chimera of the TE encoded by a FatB1 gene comprising a segment of the TE encoded by a FatB2 gene or a chimera of the TE encoded by a FatB2 gene comprising a segment of the TE encoded by a FatB1 gene.
AA. A method of altering the specificity of a plant acyl-ACP TE for at least one of its substrates, which method comprises introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the specificity of the plant acyl-ACP TE for at least one of its substrates is altered.
AB. The method of AA, which comprises mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AC. The method of AB, which further comprises mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AD. The method of AA, which further comprises altering the level of activity of the plant acyl-ACP TE by a method comprising mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the level of activity of the plant acyl-ACP TE is altered.
AE. The method of AB, which further comprises altering the level of activity of the plant acyl-ACP TE by a method comprising mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the level of activity of the plant acyl-ACP TE is altered.
AF. The method of AC, which further comprises altering the level of activity of the plant acyl-ACP TE by a method comprising mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the level of activity of the plant acyl-ACP TE is altered.
AG. A method of altering the level of activity of a plant acyl-ACP TE, which method comprises mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the level of activity of the plant acyl-ACP TE is altered.
AH. The method of AG, which further comprises altering the specificity of the plant acyl-ACP TE for at least one of its substrates by a method comprising introducing into the plant acyl-ACP TE a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, whereupon the specificity of the plant acyl-ACP TE for at least one of its substrates is altered.
AI. The method of AH, which comprises mutating at least one amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AJ. The method of AI, which further comprises mutating at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AK. An isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, wherein the isolated or purified nucleic acid molecule can be a vector.
AL. The isolated or purified nucleic acid molecule of AK, wherein the mutant plant acyl-ACP TE comprises a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AM. The isolated or purified nucleic acid molecule of AL, wherein the mutant plant acyl-ACP TE further comprises a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AN. The isolated or purified nucleic acid molecule of AK-AM, wherein the mutant plant acyl-ACP TE further comprises a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AO. An isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant plant acyl-ACP TE, which comprises a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2, wherein the isolated or purified nucleic acid molecule can be a vector.
AP. The isolated or purified nucleic acid molecule of AO, wherein the mutant plant acyl-ACP TE further comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AQ. The isolated or purified nucleic acid molecule of AP, wherein the mutant plant acyl-ACP TE comprises a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AR. The isolated or purified nucleic acid molecule of AQ, wherein the mutant plant acyl-ACP TE further comprises a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AS. A host cell or organism comprising the isolated or purified nucleic acid molecule of AK-AR.
AT. An isolated or purified mutant plant acyl-ACP TE, which comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 and/or amino acids 73-85 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AU. The isolated or purified mutant plant acyl-ACP TE of AT, which comprises a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AV. The isolated or purified mutant plant acyl-ACP TE of AU, which further comprises a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AW. The isolated or purified mutant plant acyl-ACP TE of AT-AV, which further comprises a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AX. An isolated or purified mutant plant acyl-ACP TE, which comprises a level of activity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 173, amino acid 176, and amino acid 205 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AY. The isolated or purified mutant plant acyl-ACP TE of claim AX, which further comprises a substrate specificity-altering mutation in the region corresponding to amino acids 118-167 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
AZ. The isolated or purified mutant plant acyl-ACP TE of claim AY, which comprises a mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 133, amino acid 139, amino acid 142, and amino acid 143 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
BA. The isolated or purified mutant plant acyl-ACP TE of claim AZ, which further comprises a substrate specificity-altering mutation of at least one amino acid corresponding to an amino acid selected from the group consisting of amino acid 110 and amino acid 184 of the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illuminate better the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. It should be understood that the illustrated embodiments are exemplary only, and should not be taken as limiting the scope of the invention.
This application is a divisional of U.S. patent application Ser. No. 15/188,965, filed Jun. 21, 2016, which is a divisional of U.S. patent application Ser. No. 14/141,327, filed Dec. 26, 2013, which is a continuation-in-part of U.S. patent application Ser. No. 13/558,323, filed Jul. 25, 2012 (issued Feb. 10, 2015, as U.S. Pat. No. 8,951,762), which claims priority to U.S. provisional patent application No. 61/512,373, which was filed Jul. 27, 2011, all of which are incorporated by reference in their entireties.
The work described herein was supported, at least in part, by The National Science Foundation under contract no. EEC0813570. Therefore, the Government of the United States of America has certain rights in the invention.
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| UniProt Accession No. C6LDQ9, created Sep. 22, 2009. |
| BLAST Sequence 40 from pre-grant Patent Publication No. US20110020883. |
| BLAST Sequence 45 from pre-grant Patent Publication No. US20110020883. |
| BLAST Sequence 43 from pre-grant Patent Publication No. US20110020883. |
| BLAST Sequence 61 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 138 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 57790 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 57790 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 182057 from pre-grant Patent Publication No. US20040031072. |
| BLAST Sequence 62889 from pre-grant Patent Publication No. US20070061916. |
| BLAST Sequence 182054 from pre-grant Patent Publication No. US20040031072. |
| BLAST Sequence 55416 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 55416 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 43584 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 43584 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 127 from pre-grant Patent Publication No. US20090293154. |
| BLAST Sequence 46539 from pre-grant Patent Publication No. US20060123505. |
| BLAST Sequence 164709 from pre-grant Patent Publication No. US20110131679. |
| BLAST Sequence 50930 from pre-grant Patent Publication No. US20060123505. |
| BLAST Sequence 164709 from pre-grant Patent Publication No. US20040123343. |
| BLAST Sequence 191821 from pre-grant Patent Publication No. US20110131679. |
| BLAST Sequence 191821 from pre-grant Patent Publication No. US20040123343. |
| BLAST Sequence 366754 from pre-grant Patent Publication No. US20090087878. |
| BLAST Sequence 366754 from pre-grant Patent Publication No. US20040214272. |
| BLAST Sequence 188364 from pre-grant Patent Publication No. US20110131679. |
| BLAST Sequence 188364 from pre-grant Patent Publication No. US20040123343. |
| BLAST Sequence 191819 from pre-grant Patent Publication No. US20110131679. |
| BLAST Sequence 191819 from pre-grant Patent Publication No. US20040123343. |
| BLAST Sequence 52665 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 52665 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 339361 from pre-grant Patent Publication No. US20090087878. |
| BLAST Sequence 339361 from pre-grant Patent Publication No. US20040214272. |
| BLAST Sequence 52516 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 52516 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 51871 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 51871 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 68212 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 68212 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 63 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 139 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 59 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 140 from pre-grant Patent Publication No. US20100151539. |
| BLAST Sequence 69747 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 69747 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 41073 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 41073 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 55067 from pre-grant Patent Publication No. US20060123505. |
| BLAST Sequence 50206 from pre-grant Patent Publication No. US20070283460. |
| BLAST Sequence 50206 from pre-grant Patent Publication No. US20040034888. |
| BLAST Sequence 184933 from pre-grant Patent Publication No. US20070044171. |
| BLAST Sequence 125 from pre-grant Patent Publication No. US20090293154. |
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| 20180094251 A1 | Apr 2018 | US |
| Number | Date | Country | |
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| 61512373 | Jul 2011 | US |
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| Parent | 15188965 | Jun 2016 | US |
| Child | 15836874 | US | |
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| Child | 15188965 | US |
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| Parent | 13558323 | Jul 2012 | US |
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