# Patent

## Title
Materials and methods for using an acyl-acyl carrier protein thioesterase and mutants and chimeras thereof in fatty acid synthesis

## Abstract

A method of increasing production of fatty acids comprising introducing into a host and expressing therein an acyl-acyl carrier protein (ACP) thioesterase (TE) from Bryantella formatexigens or a mutant thereof; a method of making a mutant B. formatexigens acyl-ACP TE; a method of making a chimeric Cuphea viscosissima acyl-ACP TE; a nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE or a chimeric Cuphea viscosissima acyl-ACP TE; a host comprising the nucleic acid molecule; a mutant acyl-ACP TE or chimeric Cuphea viscosissima acyl-ACP TE; a method of altering the specificity of a plant acyl-ACP TE for at least one of its substrates comprising introducing into the plant acyl-ACP TE a substrate specificity-altering mutation; and a method of altering the level of activity of a plant acyl-ACP TE.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of U.S. patent application Ser. No. 13/558,323, which was filed Jul. 25, 2012, and was published Jan. 31, 2013, as U.S. Pat. App. Pub. No. 2013/0029387, and 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.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
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.


TECHNICAL FIELD
The present disclosure relates to enzymes, mutants and chimeras thereof, fatty acid synthesis, nucleic acids, proteins and host cells and organisms.
BACKGROUND
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 (www.enzyme.cbirc.iastate.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.
SUMMARY
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).



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a sequence alignment of deduced amino acid sequences of C. nucifera (Cn) (CnFatB2 [SEQ ID NO: 4], CnFatB1 [SEQ ID NO: 5], and CnFatB3 [SEQ ID NO: 6]) and C. viscosissima (Cv) (CvFatB1 [SEQ ID NO: 2], CvFatB2 [SEQ ID NO: 3], and CvFatB3 [SEQ ID NO: 1]) acyl-ACP TEs. The putative N-terminal amino acid residue of the mature protein is leucine (▾). Two arrows indicate the conserved regions from which the degenerated primers were designed. The N-terminal sequence of CvFatB2 is incomplete (*).
FIG. 2a is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TE from C. viscosissima or C. palustris (Subfamily A). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 2b is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TE from U. Americana, I. germanica, or S. bicolor (Subfamily A). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 2c is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TEs from C. nucifera or E. guineensis (Subfamily A). In parentheses is the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 2d is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TEs from P. patens (Subfamily B), S. bicolor (Subfamily B), or M. pusilla (Subfamily D). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 3a is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TEs from B. fragilis, P. distasonis, or B. thetaiotaomicron (Subfamily F). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 3b is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TEs from S. dysgalactiae, L. brevis, or L. plantarum (Subfamily J). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 3c is a bar graph showing the fatty acid composition of E. coli K27 cultures expressing acyl-ACP TEs from A. tetradius, B. Bacteriovorus, or C. thermocellum (non-grouped). In parentheses is the organism for each sequence. Error bars represent standard errors.
FIG. 3d is a bar graph showing the fatty acid composition of an E. coli K27 culture expressing C. perfringens (Subfamily G), C. asparagiforme (Subfamily H), Geobacillus sp. (Subfamily I), D. vulgaris (Subfamily E), or B. formatexigens (Subfamily H). In parentheses are the organism and the subfamily to which each sequence belongs. Error bars represent standard errors.
FIG. 4a is a gas chromatogram of an extract from E. coli K27 culture expressing a bacterial TE (Bdellovibrio bacteriovorus, GenBank:CAE80300).
FIG. 4b is a gas chromatogram of the 2-tridecanone standard.
FIG. 4c is a gas chromatogram of a mixture of the extract of FIG. 4a and the standard of FIG. 4b. 
FIG. 4d is a mass spectrum of 2-tridecanone.
FIG. 5 is a hierarchical clustering dendrogram of acyl-ACP TEs. The acyl-ACP TEs are identified by their GenBank Accessions. Cluster analysis was performed with fatty acid composition data using Euclidean distances and Ward's hierarchical clustering method. The p-values were calculated via multiscale bootstrap resampling with 1,000 replicates. The subfamily to which each sequence belongs is indicated in parentheses. Non-grouped sequences are indicated by asterisks.
FIG. 6 is a bar graph showing total activity of wild-type acyl-ACP thioesterase from Bryantella formatexigens (EET61113) and mutants thereof. The data of all mutants (blue bars) were from single analyses, while the activity of wild-type was the average of 16 replicates.
FIG. 7 is a sequence alignment of deduced amino acid sequences of C. viscosissima (Cv) (CvFatB1 [amino acids 107-408 of SEQ ID NO: 2], CvFatB2 [amino acids 103-404 of SEQ ID NO: 3] and the strategy to generate chimeric TEs. Arrows indicate the positions where the primers were designed. Sequences for these primers are listed in Table 2. The fragments I-VI are defined by the amino acid numbers in parentheses.
FIG. 8a shows the mole percentage of individual fatty acids (FAs) from 4:0 to 16:1 as well as the total FAs (μM) for each of six chimeric TEs (i.e., rTE3, rTE12, rTE48, rTE15, rTE51, and rTE60) and C. viscosissima FatB1 (CvB1) and FatB2 (CvB2).
FIG. 8b shows the mole percentage of individual fatty acids (FAs) from 4:0 to 16:1 as well as the total FAs (μM) for each of 13 chimeric TEs (i.e., rTE4, rTE8, rTE16, rTE20, rTE24, rTE28, rTE32, rTE36, rTE40, rTE44, rTE52, rTE56 and rTE60) and C. viscosissima FatB1 (CvB1).
FIG. 9 lists the amino acid (single letter code) present at a given position in TE for the indicated organism.
FIG. 10 shows the fatty acid profiles of TE mutants on the right, wherein the green color highlights the major fatty acids produced by the TEs, the cluster analysis of the fatty acid profiles on the left, and the point mutations in each TE mutant in the middle. The wild-type enzyme is CvFatB2, which is shown with light stippling. Heavy stippling indicates residues that have been mutated to the amino acid present at the corresponding position in CvFatB1. Cross-hatching indicates residues that have been mutated to amino acids that are not present in CvFatB1 or CvFatB2.
FIG. 11 is a multiple sequence alignment of the six parental acyl-ACP TEs CnFatB2 [SEQ ID NO: 25], CnFatB3 [SEQ ID NO: 26], UaFatB1 [SEQ ID NO: 27], CpFatB1 [SEQ ID NO: 28], CvFatB1 [SEQ ID NO: 29], and CvFatB2 [SEQ ID NO: 30].
FIG. 12 is a schematic diagram of the TE variant in the pUCHisGm vector used in Example 12.
FIG. 13 is a sequence alignment of the TE variant TEGm162 [SEQ ID NO: 31] with mature CvFatB2 [SEQ ID NO: 32].
FIG. 14 is a bar graph showing the fatty acid production of parental TEs (the first five bars and the 40th bar) and TE variants. Bars represent the total fatty acids in μM, and dots represent the total fatty acids in mg/L.
FIG. 15a is a phylogenetic tree showing the relationship between parental TEs and the mutant TEs generated in Example 12 and analyzed in Example 13.
FIG. 15b is a table showing the fatty acid profiles of the parental TEs and the mutant TEs generated in Example 12 and analyzed in Example 13.
FIG. 16 is a sequence alignment of TE variants TEGm258 [SEQ ID NO: 33] and TEG205 [SEQ ID NO: 34]. The region involved in substrate specificity is indicated with a line, with dots indicating residues that have been proven to impact substrate specificity and squares indicating residues that are implicated in substrate specificity.
FIG. 17 is a sequence alignment of a 50-amino acid portion of 27 representative acyl-ACP TE sequences, including both plant and bacterial TEs that were previously functionally characterized. Two active-site motifs are indicated by lines. Arrowheads indicate previously proposed catalytic residues (N311, H313, and C348 relative to the sequence of CvFatB2). Other conserved residues (D309, Y319, and E347) are indicated by asterisks.



DETAILED DESCRIPTION
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 FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (such as family TE14), in particular an acyl-ACP TE from a bacterium, e.g., an acyl-ACP TE from Bryantella formatexigens. By “short-chain fatty acids” are meant fatty acids containing six carbons or less. By “increasing production” is meant an increase in the production of fatty acids (such as short-chain fatty acids and/or fatty acids having from about six to about 12 carbon atoms, e.g., fatty acids having from about 10 to about 12 carbon atoms, and the like) such as by at least about 5% (or 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or even greater) as compared to the 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, such as fatty acids having from about 10 to about 12 carbon atoms, and the like, in the wild-type host organism. An increase in the production of one class 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, such as fatty acids having from about 10 to about 12 carbon atoms, may or may not be reflected in an increase in the overall production of fatty acids. For example, an increase in the production of one class of fatty acids, such as short-chain fatty acids (e.g., fatty acids having less than about six carbons), may be accompanied by a decrease in the production of fatty acids of other chain lengths, such as medium chain lengths or long chain lengths. In this regard, the introduction into the host cell or organism and expression therein of a nucleic acid comprising a nucleotide sequence encoding a different TE, such as one described herein (see, e.g., FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (e.g., family TE14)), can result in an increase in the production of fatty acids containing, for example, six, eight, 10, 12, 14, 16, or more carbons.
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 FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (e.g., family TE14), in particular an acyl-ACP TE from a bacterium, e.g., an acyl-ACP TE from Bryantella formatexigens. The mutant acyl-ACP TE produces more fatty acids, such as short-chain fatty acids, in the host cell or organism than the corresponding wild-type (also referred to as “native”) acyl-ACP TE. “Short-chain fatty acids” and “increasing production” are as described above. Preferably, the mutant acyl-ACP TE derived from wild-type Bryantella formatexigens acyl-ACP TE differs from wild-type Bryantella formatexigens acyl-ACP TE by 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 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., FIG. 5, FIG. 9, Table 1, Table 2, and www.enzyme.cbirc.iastate.edu (e.g., family TE14), which website is hereby incorporated by reference for its teachings regarding acyl-ACP TEs) can serve as the source of the segment that is used to replace the segment of the wild-type Cuphea viscosissima acyl-ACP TE. Preferably, the acyl-ACP TE gene is another Cuphea viscosissima acyl-ACP TE. The method can, and preferably does, comprise replacing a segment of the CvFatB1 gene with a segment of another acyl-ACP TE gene to produce a chimeric CvFatB1 gene or replacing a segment of a CvFatB2 gene with a segment of another acyl-ACP TE gene to produce a chimeric CvFatB2 gene. In this regard, the method can comprise replacing a segment of a wild-type CvFatB1 gene with a segment of a CvFatB2 gene or replacing a segment of a wild-type CvFatB2 gene with a segment of a CvFatB1 gene. Preferably, and even desirably, the chimeric Cuphea viscosissima acyl-ACP TE encodes a chimeric acyl-ACP TE, which (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 total amount of fatty acids, both of which differ from the corresponding wild-type Cuphea viscosissima acyl-ACP TE. For example, substrate specificity can be changed from a broad-range specificity, a C8 specificity, or a C14/C16 specificity to one of the others or a specificity for a different chain length, such as C4, C6, C8, C10, C12, C14, or C16 (see, e.g., FIG. 5 and Table 2). A difference in substrate specificity can result in the production of a fatty acid not produced by the wild-type TE or the absence of production of a fatty acid produced by the wild-type TE. Activity levels by the chimeric TE, such as the total amount of fatty acids produced, can be increased or decreased compared to activity levels by the corresponding wild-type TE. Preferably, activity levels are increased.







TABLE 1





Acyl-ACP TEs







BACTERIA










Organism
GenBank ID No.
Ref Seq
Uni Prot





Acetivibrio cellulolyticus CD2

ZP_09465585.1


Acetobacterium woodii (strain ATCC
AFA47597.1
YP_005268486.1
H6LB45


29683/DSM 1030/JCM 2381/KCTC


1655)


Akkermansia muciniphila (strain ATCC
ACD05543.1
YP_001878324.1
B2UMI7


BAA-835)


Alistipes finegoldii DSM 17242
AFL79015.1
YP_006411700.1


Alistipes shahii WAL 8301
CBK64996.1

D4IPY4


Alistipes sp. HGB5
EFR57355.1
ZP_08514792.1
E4MCC6


Alkaliphilus oremlandii (strain OhlLAs)
ABW18441.1
YP_001512437.1
A8MEW2


(Clostridium oremlandii strain OhlLAs)


Anaeromyxobacter dehalogenans
ACL67544.1
YP_002494610.1
B8JAP5


(strain 2CP-1/ATCC BAA-258)


Anaeromyxobacter sp. (strain Fw109-5)
ABS24558.1
YP_001377542.1
A7H762


Anaeromyxobacter sp. (strain K)
ACG75405.1
YP_002136534.1
B4UHZ1


Anaerophaga sp. HS1

ZP_09483366.1


Anaerophaga thermohalophila DSM

ZP_08844891.1


12881


Anaerostipes sp. 3_2_56FAA
EFV23220.1
ZP_07930626.1
E5VSK1


Arcobacter nitrofigilis (strain ATCC
ADG93695.1
YP_003656202.1
D5V083


33309/DSM 7299/LMG 7604/NCTC


12251/CI) (Campylobacter nitrofigilis)


Arcobacter sp. L.
BAK73293.1
YP_005553610.1
G2HVZ0


Atopobium parvulum ATCC
ACV51065.1
YP_003159656.1
C8WAC7


33793/DSM 20469/JCM 10300/VPI


0546) (Streptococcus parvulus)


(Peptostreptococcus parvulus)


Bacillus coagulans (strain 2-6)
AEH52240.1
YP_004567626.1
F7Z1I0


Bacteroides clarus YIT 12056
EGF49737.1
ZP_08298226.1
F3PL79


Bacteroides coprosuis DSM 18011
EGJ70302.1
ZP_08457284.1
F3ZP75


Bacteroides dorei 5_1_36/D4
EEB23554.1
ZP_03302607.1
B6W3R2



EEO44480.1
ZP_08794675.1
C3R613


Bacteroides eggerthii 1_2_48FAA
EFV30016.1
ZP_07934757.1
E5WYK7


Bacteroides faecis MAJ27

ZP_09861604.1


Bacteroides fluxus YIT 12057
EGF58556.1
ZP_08299596.1
F3PR15


Bacteroides fragilis (strain YCH46)
BAD50502.1
YP_101036.1
Q64PS9


Bacteroides fragilis 3_1_12
EFR55122.1
ZP_07811188.1
E4W011


Bacteroides helcogenes (strain ATCC
ADV45243.1
YP_004162829.1
E6ST39


35417/DSM/20613/JCM 6297/P 36-108)


Bacteroides ovatus SD CC 2a
EFF59010.1
ZP_06721668.1
D4WRN0


Bacteroides salanitronis (strain DSM
ADY35429.1
YP_004257902.1
F0R2N4


18170/JCM 13567/BL78)


Bacteroides sp. 1_1_14
EFI05067.1
ZP_06994617.1
D7IBR2


Bacteroides sp. 20_3
EEY82391.1
ZP_05286609.1
D0TGY7



EFK61224.1
ZP_06076697.1
E1YZ05




ZP_07217422.1


Bacteroides sp. 3_1_19
EFI08712.1
ZP_06985974.1
D7IRY5


Bacteroides sp. 3_1_23
EEO56440.1
ZP_04550578.1
C3QWV6



EFI38044.1
ZP_07041038.1
D7K661


Bacteroides sp. 3_1_33FAA
EEO60747.1
ZP_04541663.1
C3PZJ8



EEZ23120.1
ZP_06086837.1
D1JYR1


Bacteroides sp. 3_1_40A
EET16328.1
ZP_07997247.1
C6Z619



EFV66622.1
ZP_08799591.1
E5UVX5


Bacteroides sp. 4_1_36
EFV26096.1
ZP_07938734.1
E5VAR7


Bacteroides sp. D22
EEO50828.1
ZP_06083560.1
C3QFU4



EEZ04805.1
ZP_06997928.1
D0TQX4



EFI15679.1
ZP_08787276.1
D7IYC1


Bacteroides thetaiotaomicron (strain
AAO77182.1
NP_810988.1
Q8A611


ATCC 29148/DSM 2079/NCTC


10582/E50/VPI-5482)


Bacteroides vulgates (strain ATCC
ABR40214.1
YP_001299836.1
A6L3F0


8482/DSM 1447/NCTC 11154)
EFG15793.1
ZP_06744316.1
D4VE79


Bacteroides xylanisolvens SD CC 1b.
EFG15675.1
ZP_06764643.1
D4VEU2


Bacteorides xylanisolvens XB1A
CBK66370.1

D6D8M5


Bilophila wadsworthia 3_1_6
EFV45711.1
ZP_07943134.1
E5Y2P2


butyrate-producing bacterium SS3/4
CBL41593.1

D7GUV1


butyrate-producing bacterium SSC/2
CBL38064.1

D4MZL9


Butyrivibrio crossotus DSM 2876
EFF67558.1
ZP_05793158.1
D4S2V3


Butyrivibrio fibrisolvens
CBK73219.1

D4IZD0


Butyrivibrio proteoclasticus (strain
ADL35342.1
YP_003831924.1
E0RY84


ATCC 51982/DSM 14932/B316)


Clostridium proteoclasticum)


Calditerrivibrio nitroreducens (strain
ADR19107.1
YP_004051270.1
E4TIX5


DSM 19672/NBRC 101217/Yu37-1)


Caldithrix abyssi DSM 13497
EHO418883.1
ZP_09550215.1
H1XX98


Capnocytophaga gingivalis ATCC 33624
EEK15190.1
ZP_04057029.1
C2M393


Capnocytophaga ochracea (strain ATCC
ACU91765.1
YP_003140326.1
C7M565


27872/DSM 7271/JCM 12966/VPI


2845) (Bacteroides ochraceus)


Capnocytophaga ochracea F0287
EFS97621.1
ZP_07866279.1
E4MRT3


Capnocytophaga sp. oral taxon 329 str.
EGJ52443.1
ZP_08450270.1
F3Y4U4


F0087


Capnocytophaga sp. oral taxon 329 str.
EGJ54408.1
ZP_08448171.1
F3XZ88


F0087


Capnocytophaga sp. oral taxon 338 str.
EGD34946.1
ZP_08201032.1
F0IDA8


F0234


Capnocytophaga sputigena ATCC 33612
EEB66819.1
ZP_03389980.1
E2MZN9


Carnobacterium maltaromaticum ATCC 35586
ZP_10276496.1


Carnobacterium sp. (strain 17-4)
AEB30554.1
YP_004375570.1
F4BN85


Cellulosilyticum lentocellum (strain
ADZ85723.1
YP_004310921.1
F2JL78


ATCC 49066/DSM 5427/NCIMB


11756/RHM5) (Clostridum lentocellum)


Cellulosilyticum lentocellum (strain
ADZ84608.1
YP_004309806.1
F2JLT2


ATCC 49066/DSM 5427/NCIMB


11756/RHM5) (Clostridium lentocellum)


Clostridium acetobutylicum (strain
AAK81514.1
NP_350174.1
F0KBI3


ATCC 824/DSM 792/JCM 1419/LMG
ADZ22635.1
YP_004638245.1
F7ZTD9


5710/VKM B-1787)
AEI32944.1
YP_005672730.1
Q97D89


Clostridium beijerinckii (strain ATCC
ABR37178.1
YP_001312134.1
A6M3J8


51743/NCIMB 8052) (Clostridium


acetobutylicum)


Clostridium beijerinckii (strain ATCC
ABR32877.1
YP_001307833.1
A6LR97


51743/NCIMB 8052) (Clostridium


acetobutylicum)


Clostridium botulinum (strain 657/Type
ACQ55199.1
YP_002864623.1
B1QMJ6


Ba4)
EDT85214.1
ZP_02618356.1
C3KWG6


Clostridium botulinum (strain Alaska
ACD52320.1
YP_001922691.1
B2V1S4


E43/Type E3)


Clostridium botulinum (strain ATCC
ABS34504.1
YP_001256092.1
A5I7Y7


19397/Type A)
ABS37570.1
YP_001385928.1
A7FZF7



CAL85172.1
YP_001389335.1


Clostridium botulinum (strain Eklund
ACD25059.1
YP_001887708.1
B2TRE2


17B/Type B)


Clostridium botulinum (strain H04402065/
ABS42332.1
YP_001392970.1
A7GJL0


Type A5)
ACA45501.1
YP_001783249.1
B1IHP0



ADG01324.1
YP_005676524.1
D5VZ84



CBZ05511.1
YP_005680239.1
E8ZRC8


Clostridium botulinum (strain
ACO86035.1
YP_002806157.1
B1QFF4


Kyoto/Type A2)
EDT80125.1
ZP_02615745.1
C1FNL6


Clostridium botulinum (strain Loch
ACA57020.1
YP_001788956.1
B1KU83


Maree/Type A3


Clostridium botulinum BKT015925
AEB74722.1
YP_004394719.1
F4A6R7


Clostridium botulinum C str. Eklund
EDS76894.1
ZP_02621951.1
B1BC83


Clostridium botulinum C str. Stockholm
EGO89051.1

F7MJC2


Clostridium botulinum D str. 1873
EES92019.1
ZP_04862128.1
C5VPS2


Clostridium botulinum E1 str. ‘BoNT E
EES49023.1
ZP_04821738.1
C5UVX9


Beluga’


Clostridium butyricum E4 str. BoNT E
EDT76365.1
ZP_02948643.1
B1QU73


BL5262
EEP52456.1
ZP_04529638.1
C4IMU3


Clostridium carboxidivorans P7
EET84629.1
ZP_05394929.1
C6Q1L2



EFG87972.1
ZP_06855226.1


Clostridium cellulolyticum (strain ATCC
ACL76790.1
YP_002506770.1
B8I625


35319/DSM 5812/JCM 6584/H10)


Clostridium cellulovorans (strain ATCC
ADL54009.1
YP_003845773.1
D9SPA7


35296/DSM 3052/OCM 3/743B)

ZP_07630281.1


Clostridium hahtewayi DSM 13479
EFC97397.1
ZP_06116072.2
D3ALA6


Clostridium novyi (strain NT)
ABK60402.1
YP_877012.1
A0PXB0


Clostridium papyrosolvens DSM 2782
EGD46508.1
ZP_08194021.1
F1TGE3


Clostridium perfringens (strain ATCC
ABG82470.1
YP_697308.1
Q0TM32


13124/NCTC 8237/Type A)


Clostridium perfringens (strain ATCC
ABG83514.1
YP_697307.1
B1BYH2


13124/NCTC 8237/Type A)
EDT13255.1
ZP_02634160.1
B1R6C7



EDT24316.1
ZP_02635484.1
Q0TM33


Clostridium perfringens (strain
ABG85584.1
YP_699873.1
Q0SPT4


SM101/Type A)


Clostridium perfringens CPE str. F4969
EDT25816.1
ZP_02640550.1
B1RJP3


Clostridium perfringens CPE str. F4969
EDT25796.1
ZP_02640551.1
B1RJP4


Clostridium perfringens F262
EIA15751.1

H7CZX6


Clostridium perfringens F262
EDS81696.1
ZP_02643956.1
B1BEU6



EDT77142.1
ZP_02863338.1
B1RTS9



EIA15750.1

H7CZX5


Clostridium phytofermentans (strain
ABX40638.1
YP_001557377.1
A9KS07


ATCC 700394/DSM 18823/ISDg)


Clostridium saccharolyticum (strain
ADL03414.1
YP_003821037.1
D9R5Y0


ATCC 35040/DSM 2544/NRCC 2533


WM1)


Clostridium saccharolyticum
CBK77093.1

D6DHB9


Clostridium sp. 7_2_43FAA
EEH99678.1
ZP_05132784.1
C1IBG7


Clostridium sp. 7_2_43FAA
EEH97843.1
ZP_05130949.1
C1I6P2


Clostridium sp. BNL1100
AEY65028.1
YP_005146833.1
H2JCC1


Clostridium sp. DL-VIII
EHI96936.1
ZP_09202343.1
G7MBI0


Clostridium sp. M62/1
EFE13917.1
ZP_06345069.2
D4C8T0


Clostridium sporogenes PA 3679
EHN13726.1

G9F4F0


Clostridium tetani (strain
AAO34771.1
NP_780834.1
Q899Q1


Massachusetts/E88)


Clostridium thermocellum (strain DSM
ABN54268.1
YP_001039461.1
A3DJY9


1313/LMG 6656/LQ8)
ADU73702.1
YP_005687153.1
C7HJ46



EEU00270.1
ZP_05430844.1
D1NHY5



EFB38222.1
ZP_06247582.1
E6UU77



EIC03340.1

H8EG82



EIC10345.1

H8ERY8


Coprococcus catus GD/7
CBK80368.1

D4J7P7


Coprococcus sp. ART55/1
CBK82356.1

D5HHV1


Cryptobacterium curtum (strain ATCC
ACU95033.1
YP_003151715.1
C7ML86


700683/DSM 15641/12-3)


Deferribacter desulfuricans (strain DSM
BAI79538.1
YP_003495294.1
D3PAB5


14783/JCM 11476/NBRC


101012/SSM1)


Denitrovibrio acetiphilus (strain DSM
ADD68769.1
YP_003504725.1
D4H1K9


12809/N2460)


Desulfatibacillum alkenivorans (strain
ACL04336.1
YP_002431804.1
B8FIU5


AK-01)


Desulfitobacterium dehalgenans ATCC
AFM00949.1
YP_006430742.1


51507


Desulfitobacterium hafniense (strain
ACL21191.1
YP_002459627.1
B8G1D8


DCB-2/DSM 10664)


Desulfitobacterium metallireducens DSM
EHC09806.1
ZP_08979048.1
G6GL79


15288


Desulfobacterium autotrophicum (strain
ACN14977.1
YP_002603141.1
C0QBW5


ATCC 49314/DSM 3382/HRM2)


Desulfococcus oleovorans (strain DSM
ABW68393.1
YP_001530470.1
A8ZWR2


6200/Hxd3)


Desulfohalobium retbaense (strain DSM
ACV69537.1
YP_003199115.1
C8X540


5692)


Desulfomicrobium baculatum (strain
ACU90117.1
YP_003158533.1
C7LNE8


DSM 4028/VKM B-1378) Desulfovibrio


baculatus)


Desulfonatronospira thiodismutans
EFI34439.1
ZP_07016503.1
D6SNW3


ASO3-1


Desulfosporosinus meridiei DSM 13257
EHC14266.1
ZP_08981281.1
G6G6K3


Desulfosporosinus orientis (strain ATCC
AET66411.1
YP_004968926.1
G7W7Y5


19365/DSM 765/NCIMB 8382/VKM B-


1628) (Desulfotomaculum orientis)


Desulfosporosinus youngiae DSM 17734
EHQ87820.1
ZP_09652397.1
H5Y1R7


Desulfotomaculum carboxydivorans
AEF93400.1
YP_004496312.1
F6B7F0


(strain DSM 14880/VKM B-2319/CO-1-


SRB)


Desulfotomaculum nigrificans DSM 574
EGB20954.1
ZP_08115620.1
F0DQC6


Desulfovibrio aespoeensis (strain ATCC
ADU63859.1
YP_004122605.1
E6VY45


700646/DSM 10631/Aspo-2)


Desulfovibrio africanus str. Walvis Bay
EGJ50649.1
YP_005052308.1
F3YXU7


Desulfovibrio desulfuricans (strain G20)
ABB38135.2
YP_387830.2
Q312L1


Desulfovibrio desulfuricans ND132
EGB14659.1
YP_005167463.1
F0JE50


Desulfovibrio salexigens (strain ATCC
ACS79414.1
YP_002990953.1
C6BRH4


14822/DSM 2638/NCIB 8403/VKM B-


1763)


Desulfovibrio sp. A2
EGY25981.1
ZP_08865364.1
G2H7B6


Desulfovibrio vulgaris (strain Miyazaki
ACL08376.1
YP_002435844.1
B8DRT9


F/DSM 19637)


Dethiosulfovibrio peptidovorans DSM
EFC90467.1
ZP_06391526.1
D2Z4D8


11002


Elusimicrobium minutum (strain Pei191)
ACC98705.1
YP_001876042.1
B2KDV9


Enterococcus casseliflavus ATCC 12755
EGC68270.1
ZP_08147098.1
F0EPP5


Enterococcus casseliflavus EC10
EEV28127.1
ZP_05644794.1
C9ATN7



EEV34451.1
ZP_05651118.1
C9CHB4


Enterococcus casseliflavus EC20
EEV37743.1
ZP_05654410.1
C9A551


Enterococcus faecalis (strain 62)
ADX78961.1
YP_005704694.1
C7CPH3



EET94418.1
ZP_05421510.1
C7U457



EEU22239.1
ZP_05501873.1
C7UJZ9



EEU64872.1
ZP_05561915.1
C7USW8



EEU70266.1
ZP_05567309.1
C7V886



EEU75370.1
ZP_05574399.1
C7VHP9



EEU76113.1
ZP_05575142.1
C7VYJ9



EEU81059.1
ZP_05580088.1
C7WCN3



EEU84199.1
ZP_05583228.1
C7WH64



EEU86888.1
ZP_05592094.1
C7WSC9



EEU92436.1
ZP_05597642.1
D4V053



EFG20655.1
ZP_06745965.1
F0PFE4


Enterococcus faecalis (strain ATCC
AEA92939.1
YP_005707309.1
F2MNV7


47077/OG1RF)


Enterococcus faecalis ATCC 29200
EEN72504.1
ZP_04437017.1
C2H016


Enterococcus faecalis DAPTO 512
EFE15755.1
ZP_06630131.1
D4EIL1



EFE20531.1
ZP_06631594.1
D4EX30



EFQ10983.1
ZP_07765336.1
E2YB09



EFQ68629.1
ZP_07768525.1
E2YJM4


Enterococcus faecalis Merz96
EEU66704.1
ZP_05563747.1
C7VQU1



EEU78784.1
ZP_05577813.1
C7WV73


Enterococcus faecalis T11
EEU89934.1
ZP_05595140.1
C7V0W5


Enterococcus faecalis T2
EET97613.1
ZP_05424705.1
C7CYL8


Enterococcus faecalis T8
EEU15993.1
ZP_05475136.1
C7UCD7



EEU27232.1
ZP_05557722.1
C7Y8I5


Enterococcus faecalis TUSoD Ef11
EFK76243.1
ZP_07108041.1
E1EUM8


Enterococcus faecalis TX0012
EFT94231.1

E6I3I6


Enterococcus faecalis TX0104
EEI11131.1
ZP_03949391.1
C0X740


Enterococcus faecalis TX0309B
EFU87020.1

E6GSL8



EFU94467.1

E6H424


Enterococcus faecalis TX1467
EFM69547.1
ZP_07551346.1
E0G5A0



EFM72961.1
ZP_07553437.1
E0GB90



EFM80076.1
ZP_07562578.1
E0GWQ0



EFM82210.1
ZP_07568717.1
E0H6A2



EFQ13284.1
ZP_07759854.1
E2Y3Z3



EFQ15104.1
ZP_07764076.1
E2YZ82



EFQ70935.1
ZP_07770885.1
E2Z4S5



EFT38179.1

E6F2Y1



EFT44150.1

E6F559



EFT47364.1

E6FHC7



EFT90826.1

E6FQ99



EFT97839.1

E6FWN8



EFU00982.1

E6G0U5



EFU02883.1

E6G9N2



EFU05122.1

E6GLN8



EFU10142.1

E6HEB6



EFU11766.1

E6HN77



EFU14622.1

E6HUS1



EFU16821.1

E6IDJ0



EFU88613.1

E6IKL5



EGG56243.1

F3R4H1


Enterococcus faecalis TX2141
EFT88049.1

E6IVY9


Enterococcus faecalis TX4000
EEN73225.1
ZP_04436471.1
C2DIA3



EFM65997.1
ZP_07556393.1
E0GJP6



EFM77239.1
ZP_07572356.1
E0HGP1



EFT40437.1

E6ESI6


Enterococcus faecium (strain Aus0004)
AFC64296.1
YP_005355414.1
C9B6S0



EAN09806.1
ZP_00603869.1
C9BR75



EEV42502.1
ZP_05659169.1
C9BZH1



EEV46437.1
ZP_05663104.1
C9C309



EEV47997.1
ZP_05664664.1
C9CD36



EEV53223.1
ZP_05669890.1
D0AP61



EEV57800.1
ZP_05674467.1
D4QR52



EEW62756.1
ZP_05831946.1
D4QYX5



EFF21534.1
ZP_06675770.1
D4RSJ8



EFF29090.1
ZP_06676257.1
D4SPZ7



EFF30890.1
ZP_06678973.1
G9SNG7



EFF35697.1
ZP_06701551.1
G9SWE6



EHM36206.1

H8L8C1



EHM36898.1

Q3Y000


Enterococcus faecium Com15
EEV62230.1
ZP_05678897.1
C9AQE0


Enterococcus faecium E1636
EEW64692.1
ZP_05923461.1
D0AHZ9



EFD09525.1
ZP_06446970.1
D3LH99



EFF23086.1
ZP_06695563.1
D4RAG0


Enterococcus faecium E1679
EFF25207.1
ZP_06699424.1
D4RLH1


Enterococcus faecium E980
EFF38835.1
ZP_06681449.1
D4QHC6


Enterococcus faecium PC4.1
EEV51369.1
ZP_05668036.1
C9AJN9



EEV60229.1
ZP_05676896.1
C9BGE2



EFF61513.1
ZP_06624194.1
D4VWH8


Enterococcus faecium TX0133a01
AFK59819.1
YP_006376801.1
E4I8N4



EFR68006.1
ZP_07846162.1
E4IJA4



EFR70537.1
ZP_07849882.1
E4INZ4



EFR75054.1
ZP_07851522.1
EVIXY0



EFR77028.1
ZP_07854658.1
EVJAY8



EFS06349.1
ZP_07859216.1
EVJI68



EFS10037.1
ZP_07861746.1


Enterococcus faecium TX1330
EEI61496.1
ZP_03980399.1
C2H7W0


Enterococcus gallinarum EG2
EEV32970.1
ZP_05649637.1
C9A053


Enterococcus italicus DSM 15952
EFU73343.1
ZP_07896506.1
E6LHJ1


Ethanoligenens harbinense (strain DSM
ADU28280.1
YP_004093011.1
E6U8G1


18485/JCM 12961/CGMCC 1.5033


YUAN-3)


Eubacterium cellulosolvens 6
EIM57815.1
ZP_10167425.1


Eubacterium limosum (strain KIST612)
ADO35661.1
YP_003958624.1
E3GJ26


Eubacterium rectale DSM 17629
CBK89551.1

D6E2B1


Eubacterium rectal M104/1
CBK94159.1

D4JKL2


Eubacterium saburreum DSM 3986
EFU75440.1
ZP_07905804.1
E6LS63


Eubacterium siraeum 70/3
CBK97519.1

D4JWQ0


Eubacterium siraeum V10Sc8a
CBL35446.1

D4MNW3


Faecalibacterium prausnitzii L2-6
CBK98400.1

D4K4F7


Fibrella aestuarina BUZ 2
CCG99613.1

I0K660


Fibrisoma limi BUZ 3
CCH55838.1


Fibrobacter succinogenes (strain ATCC
ACX75214.1
YP_003249696.1
C9RRM8


19169/S85)
ADL24713.1
YP_005822012.1


Finegoldia magna (strain ATCC 29328)
BAG08537.1
YP_001692427.1
B0S2E7


(Peptostreptococcus magnus)


Finegoldia magna
EFL54101.1
ZP_07321135.1
E1KXS7


SY403409CC001050417
EGS34728.1

F9MZ32


Flavobacterium columnare (strain
AEW86359.1
YP_004942152.1
G8X6Z8


ATCC 49512/CIP 103533/TG 44/87)


Flavobacterium frigoris PS1
EIA08417.1
ZP_09895618.1
H7FRY9


Flavobacterium johnsoniae (Strain
ABQ06468.1
YP_001195787.1
A5FE97


ATCC 17061/DSM 2064/UW101)


Cytophaga johnsonae


Flavonifractor plautii ATCC 29863
EHM53486.1
ZP_09383254.1
G9YNF5


Flexistipes sinusarabici (strain DSM
AEI14379.1
YP_004602947.1
F8E426


4947/MAS 10)


Fructobacillus fructosus KCTC 3544

ZP_08660317.1


Geobacillus sp. (strain Y412MC10)
ACX63219.1
YP_003241026.1
D3EF08


Geobacter lovleyi (Strain ATCC BAA-
ACD96083.1
YP_001952603.1
B3E596


1151/DSM 17278/SZ)


Geobacter metallireducens (strain GS-
ABB31963.1
YP_384688.1
H1L7J4


15/ATCC 53774/DSM 7210)
EHP86324.1

Q39UW1


Geobacter sp. (strain FRC-32)
ACM20973.1
YP_002538074.1
B9M0X0


Granulicatella adiacens ATCC 49175
EEW37696.1
ZP_05737219.1
C8NEY7


Granulicatella elegans ATCC 700633
EEW93633.1
ZP_05851687.1
D0BKN0


Haliscomenobacter hydrossis (strain
AEE52567.1
YP_004449440.1
F4KWT4


ATCC 27775/DSM 1100/LMG 10767 O)


Haloplasma contractile SSD-17B
EGM25295.1
ZP_08557504.1
F7Q2G6



EGM26151.1
ZP_08558128.1


Jonquetella anthropi DSM 22815
EHM12447.1

H0ULI2


Jonquetella anthropi E3_33 E1
EEX49185.1
ZP_05859848.1
C9M5Q8


Lachnospiraceae bacterium 5_1_63FAA
EFV17918.1
ZP_07955084.1
E5VG94


Lachnospiraceae bacterium oral taxon
EHO50163.1
ZP_09561948.1
H1LYH8


082 str. F0431


Lactobacillus acidipiscis KCTC 13900

ZP_09456326.1


Lactobacillus acidophilus (strain 30SC)
ADZ06571.1
YP_004286708.1
F0TIR4


Lactobacillus aciophilus (strain ATCC
AAV42277.1
YP_193308.1
Q5FLZ7


700396/NCK56/N2/NCFM)


Lactobacillus acidophilus ATCC 4796
EEJ76335.1
ZP_04021090.1
C2HMJ5


Lactobacillus amylolyticus DSM 11664
EFG55410.1
ZP_06818559.1
D4YTY9


Lactobacillus amylovorus (strain GRL
ADQ58392.1
YP_004031187.1
E4SLD4


1118)
AEA31360.1
YP_005853365.1
F2M221


Lactobacillus animalis KCTC 3501

ZP_08549496.1


Lactobacillus brevis (strain ATCC
ABJ63754.1
YP_794785.1
Q03SR8


367/JCM 1170)


Lactobacillus brevis subsp. gravesensis
EEI69866.1
ZP_03940727.1
C2D5L9


ATCC 27305


Lactobacillus buchneri (strain NRRL B-
AEB73621.1
YP_004398684.1
F4FX93


30929)


Lactabacillus buchneri ATCC 11577
EEI18325.1
ZP_03943743.1
C0WUL3


Lactobacillus casei (strain ATCC 334)
ABJ70998.1
YP_003789283.1
D8GB52



ADK19433.1
YP_005857330.1
F2MA80



AEA54746.1
YP_005860469.1
F2MJ21



AEA57928.1
YP_807440.1
Q035X4


Lactobacillus coleohominis 101-4-CHN
EEU30164.1
ZP_05553571.1
C7XWA4


Lactobacillus crispatus (strain ST1)
CBL49834.1
YP_003600859.1
D5H1E9


Lactobacillus crispatus 125-2-CHN
EEU18812.1
ZP_05550017.1
C7XLF9


Lactobacillus crispatus CTV-05
EEJ70241.1
ZP_03995680.1
C2KCV6



EEX28703.1
ZP_06020517.1
D0DJR2



EFE00376.1
ZP_06626062.1
D4FBS4



EFQ44288.1
ZP_07789659.1
E3R4N0


Lactobacillus crispatus MV-1A-US
EEU28844.1
ZP_05554513.1
C7Y3T1


Lactobacillus delbrueckii subsp.
ADY85572.1
YP_005852596.1
F0JZ70


bulgaricus (strain 2038)


Lactobacillus delbrueckii subsp.
ABJ58999.1
YP_813437.1
Q048X3


bulgaricus (strain ATCC BAA-365)


Lactobacillus delbrueckii subsp.
ADQ61561.1
YP_004034538.1
E4SWA3


bulgaricus (strain ND02)


Lactobacillus delbrueckii subsp.
EFK32863.1
ZP_07091661.1
D8FL81


bulgaricus PB2003/044-T3-4


Lactobacillus delbrueckii subsp. lactis
EGD27772.1

F0HUG7


DSM 20072


Lactobacillus farciminis KCTC 3681

ZP_08576095.1


Lactobacillus fermentum (strain CECT
ADJ40858.1
YP_005848352.1
D8IGS7


5716)


Lactobacillus fructivorans KCTC 3543

ZP_08652157.1


Lactobacillus gasseri (strain ATCC
ABJ59782.1
YP_814220.1
Q045U0


33323/DSM 20243)


Lactobacillus gasseri 202-4
EEQ26038.1
ZP_04643995.1
C4VSR8


Lactobacillus gasseri 224-1
EFB62115.1
ZP_06262095.1
D1YK76


Lactobacillus gasseri JV-V03
EFJ69165.1
ZP_07058478.1
D7V535


Lactobacillus gastricus PS3
EHS87218.1
ZP_09786106.1
H4GIM3


Lactobacillus helveticus (strain H10)
ADX69593.1
YP_005849580.1
F0NV99


Lactobacillus helveticus MTCC 5463
EEW67886.1
ZP_05752683.1
C9M2C2



EGF34731.1

F3MQC6


Lactobacillus hilgardiiATCC 8290
EEI25650.1
ZP_03952565.1
C0XG64


Lactobacillus iners AB-1

ZP-07267176.1


Lactobacillus iners ATCC 55195
EEW51705.1
ZP_05744259.1
C8PCP7



EFO66686.1
ZP_07697641.1
E1NFD0



EFQ50621.1
ZP_07732239.1
E3BYM9



EFU79110.1
ZP_07906033.1
E6LSU2


Lactobacillus iners LactinV 09V1-c
EFO67514.1
ZP_07699507.1
E1NKQ9


Lactobacillus iners LEAF 3008A-a
EFQ51957.1
ZP_07731033.1
E3C233


Lactobacillus iners SPIN 1401G
EFO69137.1
ZP_07700645.1
E1NP18



EGG31776.1
ZP_08277548.1
F3M0Y4


Lactobacillus iners SPIN 2503V10-D
EFO71603.1
ZP_07703903.1
E1NYD0


Lactobacillus iners UPII 143-D
EFQ47178.1
ZP_07733650.1
E3BU75



EFQ49253.1
ZP_07735732.1
E3BVE7



EGC79722.1
ZP_08173986.1
F0GLZ3


Lactobacillus iners UPII 60-B
EGC80907.1
ZP_08175329.1
F0GQU0


Lactobacillus jensenii 208-1
EEQ24340.1
ZP_04645666.1
C4VMZ1



EEX27386.1
ZP_05865781.1
D0DNZ9



EFA93633.1
ZP_06339831.1
D1WHH0


Lactobacillus johnsonii DPC 6026
AEB92746.1
YP_005861696.1
F4AEU9


Lactobacillus johnsonii pf01
EGP13921.1

F7SCN1


Lactobacillus kefiranofaciens (strain
AEG39778.1
YP_004561880.1
F6CD69


ZW3)


Lactobacillus kisonensis F0435
EHO52285.1
ZP_09555400.1
H1LET0


Lactobacillus malefermentans KCTC

ZP_09441303.1


3548


Lactobacillus mali KCTC 3596 = DSM

ZP_09447064.1


20444


Lactobacillus mucosae LM1
EHT15844.1
ZP_09815086.1
H3RQN4


Lactobacillus oris F0423
EGS36091.1

F9JK82


Lactobacillus oris PB013-T2-3
EFQ53035.1
ZP_07729836.1
E3C864


Lactobacillus paracasei subsp. paracasei
EEI68786.1
ZP_03963784.1
C2FBR3


8700:2
EEQ66724.1
ZP_04673142.1
C5F8Y0


Lactobacillus parafarraginis F0439
EHL96655.1
ZP_09393527.1
G9ZRF4


Lactobacillus plantarum (strain ATCC
ADN97761.1
YP_003923855.1
D7VG69


BAA-793/NCIMB 8826/WCFS1)
CCC78182.1
YP_004888696.1
E1TKY4



EFK27869.1
ZP_07079657.1
F9ULU3



EHS83236.1

H3P172


Lactobacillus reuteri (strain ATCC
AEI57752.1
YP_004650042.1
C2GRC8


55730/SD2112)
EEI64955.1
ZP_03975201.1
F8DLS3


Lactobacillus reuteri 100-23
EDX41870.1
ZP_03074105.1
B3XQR9


Lactobacillus rhamnosus (strain ATCC
BAI42696.1
YP_003172003.1
B5QQB5


53103/GG)
CAR88152.1
YP_005866661.1
C7TEV9



EDY98171.1
ZP_03212443.1
G6AK63



EHJ21134.1

G6JOH2



EHJ36340.1


Lactobacillus rhamnosus (strain Lc 705)
AER64996.1
YP_003174934.1
C2JZ75



CAR91083.1
YP_005873849.1
C7TLJ9



EEN79670.1
ZP_04441678.1
G7UXI5


Lactobacillus ruminis (strain ATCC
AEN77890.1
YP_004831826.1
G2SMB8


27782/RF3)


Lactobacillus ruminis SPM0211
EFZ34938.1
ZP_08080403.1
E7FPK7



EGM50173.1
ZP_08564070.1
F7R2D3



EGX99156.1


Lactobacillus salivarius (strain CECT
ADJ79282.1
YP_005864069.1
D8ILY0


5713)


Lactobacillus salivarius (strain UCC118)
ABE00028.1
YP_536111.1
C2EGC7



EEJ74495.1
ZP_04009005.1
Q1WSV1


Lactobacillus salivarius ACS-116-V-
EFK79641.1
ZP_07206660.1
E1JN74


Col5a


Lactobacillus salvarius GJ-24
EGL99750.1

F5VCC4



EGM52363.1

F7QU96


Lactobacillus salivarius SMXD51
EIA32146.1

H7G0I9


Lactobacillus vaginalis ATCC 49540
EEJ40900.1
ZP_03959573.1
C2ETB7


Lactobacillus versmoldensis KCTC 3814

ZP_09443147.1


Lactobacillus zeae KCTC 3804

ZP_09454130.1


Lactococcus garvieae IPLA 31405
EIT66252.1


Lactococcus garvieae (strain Lg2)
BAK58395.1
YP_004779059.1
F9V8F4


(Enterococcus seriolicida)
BAK60363.1
YP_005870647.1
F9VDG2


Lactococcus lactis subsp. cremoris (strain
ABJ72781.1
YP_809203.1
Q02Z41


SK11)


Lactococcus lactis subsp. cremoris A76
AEU40439.1
YP_005875862.1
G8P2V5


Lactococcus lactis subsp. lactis (strain
ADZ63759.1
YP_005868493.1
F2HJJ6


CV56)
EHE92562.1

G6FES7


Lactococcus lactis subsp. lactis IO-1
BAL51042.1

H5SYX8


Leuconostoc citreum (strain KM20)
ACA83334.1
YP_001728778.1
B1MVT0



CCF27846.1

H1WZU7


Leuconostoc citreum LBAE C10
CCF23736.1

H1WN37


Leuconostoc citreum LBAE C11
CCF26033.1

H1WUN4


Leuconostoc fallax KCTC 3537

ZP_08313133.1


Leuconostoc mesenteroides subsp.
ABJ61410.1
YP_005173661.1
G7VJW8


mesenteroides (strain ATCC 8293/NCDO
AET29695.1
YP_817783.1
Q03ZG2


523)


Leuconostoc pseudomesenteroides KCTC

ZP_08658910.1


3652


Leuconostoc pseudomesenteroides KCTC

ZP_08657880.1


3652


Marvinbryantia formatexigens DSM
EET58368.1
ZP_05348824.1
C6LLV8


14469


Melissococcus plutonius (strain ATCC
BAK22205.1
YP_004457014.1
F3YCH7


35311/CIP 104052/LMG 20360/NCIMB


702443)


Melissococcus plutonius (strain
BAL61565.1
YP_005319060.1
H5T322


DAT561)


Mesotoga prima MesG1.Ag.4.2
AFK07784.1
YP_006347027.1


Microscilla marina ATCC 23134
EAY28464.1
ZP_01690629.1
A1ZMT4


Moorella thermoacetica (strain ATCC
ABC20493.1
YP_431036.1
Q2RGE6


39073)


Myroides odoratus DSM 2801
EHQ43781.1
ZP_09673466.1
H1ZDF8


Odoribacter splanchnicus (strain ATCC
ADY34476.1
YP_004254656.1
F9ZBU7


29572/DSM 20712/JCM 15291/NCTC


10825/1651/6) (Bacteroides


splanchnicus)


Oenococcus kitaharae DSM 17330
EHN59872.1

G9WGP5


Olsenella uli (strain ATCC 49627/DSM
ADK67921.1
YP_003800801.1
E1QZV7


7084/CIP 109912/JCM 12494/VPI


D76D-27C) (Lactobacillus uli)


Opitutus terrae (strain DSM
ACB75273.1
YP_001817873.1
B1ZXQ1


11246/PB90-1)


Ornithinibacillus scapharcae TW25

ZP_08784004.1


Paenibacillus dendritiformis C454
EHQ61755.1
ZP_09676931.1
H3SGM0


Paenibacillus lactis 154
EHB50198.1
ZP_09004493.1
G4HNN3


Paenibacillus sp. HGF5
EGG35794.1
ZP_08280757.1
F3MA61


Paenibacillus vortex V453
EFU42569.1
ZP_07898346.1
E5YSB9


Paludibacter propionicigenes (strain
ADQ78301.1
YP_004041286.1
E4T0E2


DSM 17365/JCM 13257/WB4)


Parabacteroides distasonis (strain ATCC
ABR43801.1
YP_001303423.1
A6LDN7


8503/DSM 20701/NCTC 11152)


Parabacteroides sp. D13
EEU50458.1
ZP_05546795.1
C7XCA8


Paraprevotella clara YIT 11840
EHH00544.1
ZP_09107765.1
G5SQZ1


Paraprevotella xylaniphila YIT 11841
EGG54116.1
ZP_08320615.1
F3QU31


Pediococcus acidilactici DSM 20284
EFL95888.1
ZP_07367805.1
E0NFJ4


Pediococcus acidilactici MA18/5M
EFA26407.1
ZP_06197399.1
D2EKP7



EHJ20298.1

G6IS05


Pediococcus pentosaceus (strain ATCC
ABJ68507.1
YP_804949.1
H8G000


25745/183-1w)
CCG89967.1

Q03E65


Pelobacter propionicus (strain DSM
ABL00830.1
YP_902887.1
A1AU09


2379)


Peptoniphilus harei ACS-146-V-Sch2b
EFR32523.1
ZP_07822609.1
E4L0C9


Prevotella bivia JCVIHMP010
EFB92152.1
ZP_06269388.1
D1Y0N5



EIM31835.1


Prevotella buccae ATCC 33574
EFU30435.1
ZP_07882963.1
E6K8D5


Prevotella buccalis ATCC 35310
EFA92524.1
ZP_06286413.1
D1W477


Prevotella dentalis DSM 3688
EGQ16303.1
ZP_08669671.1
F9D1E1


Prevotella dentalis DSM 3688
EHO56109.1

H1MBA2


Prevotella denticola (strain F0289)
AEA20475.1
YP_004329120.1
F2KTP5


Prevotella denticola CRIS 18C-A
EGC84969.1
ZP_08173659.1
F0HB58


Prevotella disiens FB035-09AN
EFL45598.1
ZP_07323756.1
E1KSA6


Prevotella intermedia 17
AFJ08475.1
YP_006299246.1


Prevotela marshii DSM 16973
EFM02212.1
ZP_07365421.1
E0NRS4


Prevotella melaninogenica (strain ATCC
ADK95990.1
YP_003814035.1
D9RUL0


25845/DSM 7089/JCM 6325/VPI


2381/B282) (Bacteroides


melaninogenicus)


Prevotella multiformis DSM 16608
EGC20653.1
ZP_08135691.1
F0F5N4


Prevotella multisaccharivorax DSM
EGN58078.1
ZP_08580508.1
F8NCP3


17128


Prevotella nigrescens ATCC 33563
EGQ15946.1
ZP_08672539.1
F9D9K9


Prevotella oralis ATCC 33269
EFZ37525.1
ZP_08084447.1
E7RP82


Prevotella oris C735
EFI48008.1
ZP_07035544.1
D7NEM6


Prevotella ruminicola (strain ATCC
ADE82257.1
YP_003574402.1
D5ERZ8


19189/JCM 8958/23)


Prevotella salivae DSM 15606
EFV04584.1
ZP_07961907.1
E6MNZ7


Prevotella sp. oral taxon 306 str. F0472
EID32261.1
ZP_09968086.1
I0T9F8


Prevotella stercorea DSM 18206
EHJ38668.1
ZP_09198687.1
G6AZ54


Pseudoramibacter alactolyticus ATCC
EFV02259.1
ZP_07920643.1
E6MF99


23263


Rhodothermus marinus (strain ATCC
ACY48916.1
YP_003291304.1
D0MKK4


43812/DSM 4252/R-10) Rhodothermus


obamensis)


Rhodothermus marinus SG0.5JP17-172
AEN72763.1
YP_004824600.1
G2SK07


Roseburia hominis (strain DSM
AEN95631.1
YP_004837563.1
G2T1Y7


16839/NCIMB 14029/A2-183)


Roseburia intestinalis L1-82
EEV02992.1
ZP_04741938.1
C7G5B5


Roseburia intestinalis XB6B4
CBL09912.1

D4KSR0



CBL12214.1

D4KXX4


Ruminococcus albus (strain ATCC
ADU24217.1
YP_004090103.1
E6UKM1


27210/DSM 20455/JCM 14654/NCDO


2250/7)


Ruminococcus obeum A2-162
CBL24676.1

D4LV68


Ruminococcus sp. SR1/5
CBL20901.1

D4LM40


Ruminococcus torques L2-14
CBL26041.1

D4M479


Salinibacter ruber (strain DSM
ABC43900.1
YP_444210.1
Q2S6H1


13855/M31)


Salinibacter ruber (strain M8)
CBH22976.1
YP_003569928.1
D5H4M1


Sphaerochaeta pleomorpha (strain ATCC
AEV29849.1
YP_005062859.1
G8QR01


BAA-1885/DSM 22778/Grapes)


Sphingobacterium spiritovorum ATCC
EEI90316.1
ZP_03970072.1
C2G3W1


33300


Sphingobacterium spiritivorum ATCC
EFK55869.1
ZP_07082740.1
D7VQ18


33861


Spirochaeta africana DSM 8902
AFG37541.1
YP_005475248.1
H9UJ48


Sprochaeta caldaria (strain ATCC
AEJ20927.1
YP_004699435.1
F8F2E5


51460/DSM 7334/H1)


Sprochaeta caldaria (strain ATCC
AEJ18974.1
YP_004697482.1
F8F0B0


51460/DSM 7334/H1)


Spirochaeta coccoides (strain ATCC
AEC02997.1
YP_004412379.1
F4GLV6


BAA-1237/DSM 17374/SPN1)


Spirochaeta coccoides (strain ATCC
AEC02807.1
YP_004412189.1
F4GJR0


BAA-1237/DSM 17374/SPN1)


Spirochaeta smaragdinae (strain DSM
ADK83104.1
YP_003805698.1
E1R9D5


11293/JCM 15392/SEBR 4228)


Spirochaeta smaragdinae (strain DSM
ADK82412.1
YP_003805006.1
E1RAP4


11293/JCM 15392/SEBR 4228)


Spirochaeta thermophila (strain ATCC
ADN01075.1
YP_003873348.1
E0RU19


49972/DSM 6192/RI 19.B1)


Spirochaeta thermophila DSM 6578
AEJ60373.1
YP_006044090.1
G0GBL4


Spirosoma linguale (strain ATCC
ADB42312.1
YP_003391111.1
D2QU30


33905/DSM 75/LMG 10896)


Streptococcus anginosus 1_2_62CV
EFW07929.1
ZP_08013620.1
E7GWK1


Streptococcus anginosus CGUG 39159
EID21884.1

I0SET1


Streptococcus anginosus F0211
EFU22895.1
ZP_07863625.1
E6IZR2


Streptococcus anginosus SK52 = DSM
EGL47971.1
ZP_08524336.1
F5TZ42


20563


Streptococcus australis ATCC 700641
EFV99485.1
ZP_08021412.1
E7SBM6



EGU64265.1


Streptococcus canis FSL Z3-227
EIQ82096.1
ZP_10274029.1


Streptococcus constellatus subsp.
EID22796.1

I0SHE3


constellatus SK53


Streptococcus constellatus subsp.
EGV09466.1
ZP_08762014.1
F9P6H6


pharyngis SK1060 = CCUG 46377


Streptococcus criceti HS-6
EHI74170.1
ZP_09123331.1
G5JTU8


Streptococcus cristatus ATCC 51100
EFX53291.1
ZP_08059281.1
E8JTL6



EGU66254.1


Streptococcus downei F0415
EFQ56787.1
ZP_07726381.1
E3CLH4


Streptococcus dysgalactiae subsp.
ADX24753.1
YP_006013333.1
E8QAX7


equisimilis (strain ATCC 12394/D166B)


Streptococcus dysgalactiae subsp.
BAH81730.1
YP_002996944.1
C5WH65


equisimilis (strain GGS_124)


Streptococcus dysgalactiae subsp.
EGL49418.1

F5U565


equismilis SK1249


Streptococcus equi subsp. zooepidemicus
AEJ25504.1

F8IMR8


(strain ATCC 35246/C74-63)


Streptococcus equi subsp. zooepidemicus
ACG62550.1
YP_002123563.1
B4U3I3


(strain MGCS10565)


Streptococcus equines ATCC 9812
EFW88979.1
ZP_08041402.1
E8JP85


Streptococcus gallolyticus subsp.
EFM29278.1
ZP_07464895.1
E0PL23


gallolyticus TX20005


Streptococcus ictaluri 707-05
EHI69863.1
ZP_09126352.1
G5K2J5


Streptococcus infantarius (strain CJ18)
AEZ62572.1
YP_005204043.1
H6PBS3


Streptococcus infantis ATCC 700779
EFX36879.1
ZP_08061125.1
E8JZ60



EIG40319.1


Streptococcus infantis SK1076
EGL85964.1
ZP_08523541.1
F5W0Y7


Streptococcus infantis SK1302
EFO55275.1
ZP_07692775.1
E1M520


Streptococcus infantis SK970
EGV04542.1

F9PUI2


Streptococcus infantis X
EGV15183.1

F9PB80


Streptococcus intermedius SK54
BAM23377.1
YP_006469625.1
I0X1J6



EID82512.1


Streptococcus macacae NCTC 11558
EHJ52132.1
ZP_09134350.1
G5JVK4


Streptococcus macedonicus (strain ACA-
CCF02741.1
YP_005095067.1
H2A7K5


DC 198)


Streptococcus mitis (strain B6)
CBJ21968.1
YP_003445836.1
D3H7X6


Streptococcus mitis ATCC 6249
EFM31215.1
ZP_07462962.1
E0PS01


Streptococcus mitis bv. 2 str. F0392
EGR93006.1

F9P0J2


Streptococcus mitis bv. 2 str. SK95
EGU67810.1

F9LW41


Streptococcus mitis NCTC 12261
EFN95948.1
ZP_07638954.1
E1M0K5


Streptococcus mitis SK1073
EGP65457.1

F9HDJ2


Streptococcus mitis SK1080
EGP70417.1

F9HK06


Streptococcus mitis SK569
EGU71416.1

F9MHA3


Streptococcus mitis SK575
EID27575.1

I0SW22


Streptococcus mitis SK579
EID31778.1

I0T825


Streptococcus mitis SK597
EFO00396.1
ZP_07641986.1
E1LSZ7


Streptococcus mitis SK616
EID24616.1

I0SML3


Streptococcus oralis SK10
EIC79352.1

I0QDK0


Streptococcus oralis SK100
EIC76735.1

I0Q633


Streptococcus oralis SK1074
EID25784.1

I0SQY1


Streptococcus oralis SK255
EGL90926.1

F5VTF6


Streptococcus oralis SK313
EGV00858.1

F9Q4H5


Streptococcus oralis SK610
EIC75997.1

I0Q3Z5


Streptococcus parasanguinis F0449
EIG27302.1


Streptococcus parasanguinis (strain
AEH55709.1
YP_004621637.1
F8DI02


ATCC 15912/DSM 6778/CIP 104372


LMG 14537)


Streptococcus parasanguinis ATCC 903
EFX38996.1
ZP_08063273.1
E8K5A8


Streptococcus parasanguinis F0405
EFQ55999.1
ZP_07726902.1
E3CB49


Streptococcus parasanguinis SK236
EGU65274.1

F9M0Q9


Streptococcus parauberis (strain KCTC
AEF25390.1
YP_004479062.1
F5ZIR6


11537)


Streptococcus parauberis NCFD 2020
EGE54640.1
ZP_08246038.1
F1Z1C3


Streptococcus peroris ATCC 700780
EFX40622.1
ZP_08065211.1
E8KB51


Streptococcus pneumoniae (strain 670-
ADM90954.1
YP_003879054.1
A5MHD1


6B)
EDK68094.1
ZP_01830980.1
E0TMA5



EGI85205.1

F3VJ08



EHD49693.1

G6KXZ1



EHD63537.1

G6M938



EHD88133.1

G6NUS3



EHE00949.1

G6QQ59



EHE60798.1

G6VMF7



EHZ08811.1

H7K7G8



EIA01765.1

H7NWY1



EIA03628.1

H7P9A4


Streptococcus pneumoniae (strain 70585)
ACO16116.1
YP_002740683.1
A5M825



EDK66086.1
ZP_01827724.1
C1C807



EHD28957.1
ZP_04524259.1
G6JBH8



EHD89063.1
ZP_04598288.1
G6PTT6



EHE10731.1

G6RMA4



EHY96087.1

H7JGM5



EHZ58538.1

H7MCK4


Streptococcus pneumoniae (strain
ACA37604.1
YP_001694843.1
A5MT27


Hungary 19A-6)
EDK82213.1
ZP_01834726.1
B1ICK8



EHD56190.1

G6LGP3



EHZ17150.1

H7I8B3



EHZ26304.1

H7KWU0



EHZ51454.1

H7LTH4


Streptococcus pneumoniae GA05245
EGI85461.1

F3WAV6



EHE02013.1

G6R2M6



EHZ03819.1

H7PKX9


Streptococcus pneumoniae GA11663
EHE76579.1

G6P9A5


Streptococcus pneumoniae GA13637
EHD92416.1

G6Q076


Streptococcus pneumoniae GA40028
EDT91262.1
ZP_02710611.1
B2DHG4



EHE29264.1

G6T4S1



EHZ39486.1

H7NP72



EHZ97713.1

H7QAJ1


Streptococcus pneumoniae GA40563
EHZ44498.1

H7LG30


Streptococcus pneumoniae GA41688
EHE25505.1

G6SS73


Streptococcus pneumoniae GA47373
EHE36709.1

G6TER8


Streptococcus pneumoniae GA47439
EDT95056.1
ZP_02715455.1
B2DT86



EHE39175.1

G6TUT3


Streptococcus pneumoniae GA47461
EHZ60517.1

H7MHZ6


Streptococcus pneumoniae GA47522
EHD39940.1

G6K6A5



EHZ64825.1

H7HCA4



EHZ94055.1

H7MPL4


Streptococcus pneumoniae GA47778
EHD60478.1

G6LTP3



EHE14708.1

G6RV07



EHE17320.1

G6RZ73



EHE42733.1

G6U6L7


Streptococcus pneumoniae GA49194
EGJ14922.1

F3XKI1



EHZ72974.1

H7J3Y0



EHZ90787.1

H7NER5


Streptococcus pneumoniae GA49542
EDK79250.1
ZP_01822626.1
A5LTH7



EDT92444.1
ZP_02713772.1
B2DPR6



EGI84194.1

F3VP30



EHD76303.1

G6NBR6



EHE37076.1

G6TMQ1



EHE51720.1

G6UNS9



EHE67173.1

G6WCE5



EHZ48988.1

H7LJZ9



EHZ76888.1

H7NIU9


Streptococcus porcinus str. Jelinkova 176
EGJ26555.1
ZP_08398558.1
F3LB38


Streptococcus pseudopneumoniae (strain
AEL10828.1
YP_004768688.1
G0IAN6


IS7493)


Streptococcus pseudopneumoniae SK674
EID23674.1
ZP_09992237.1
I0SJX1



EID70047.1

I0W0Y1


Streptococcus pseudoporcinus LQ 940-
EFR44687.1
ZP_07823857.1
E4L3R5


04
EHI65004.1

G5KAN5


Streptococcus pyogenes HKU
EIK42042.1


QMH11M0907901


Streptococcus pyogenes Alab49
AEQ24391.1
YP_006071945.1
G4R4C7


Streptococcus pyogenes ATCC 10782
EFM33381.1
ZP_07460727.1
E0PWA2


Streptococcus pyogenes MGAS1882
AFC66139.1
YP_005388837.1
H8HAK4



AFC68003.1
YP_005411534.1
H8HE06


Streptococcus pyogenes serotype M1
AAZ51384.1
YP_282129.1
Q48Z34


Streptococcus pyogenes serotype M12
ABF32069.1
YP_596613.1
Q1JC16


(strain MGAS9429)
ABF35892.1
YP_600436.1
Q1JM00


Streptococcus pyogenes serotype M2
ABF33948.1
YP_598492.1
Q1JH46


(strain MGAS10270)


Streptococcus pyogenes serotype M28
AAX71858.1
YP_280213.1
Q48TU8


Streptococcus pyogenes serotype M4
ABF37868.1
YP_602412.1
Q1J6W5


(strain MGAS10750)


Streptococcus pyogenes serotype M6
AAT86926.1
YP_060109.1
Q5XCD7


Streptococcus salivarius (strain 57.1)
AEJ53614.1
YP_006068477.1
F8HDR2



EGX30167.1

G2GSH1


Streptococcus salivarius SK126
EEK10550.1
ZP_04061476.1
C2LQT3


Streptococcus sanguinis ATCC 49296
EFU62475.1
ZP_07888343.1
E6KNA8


Streptococcus sanguinis SK1056
EGJ39149.1

F3UB48


Streptococcus sanguinis SK1057
EGF06788.1

F2BSB3


Streptococcus sanguinis SK1058
EGF21443.1

F2CLJ1


Streptococcus sanguinis SK1087
EGG40307.1

F3SHX4


Streptococcus sanguinis SK115
EGD31794.1

F0I7V9


Streptococcus sanguinis SK150
EGD36508.1

F0IMN8


Streptococcus sanguinis SK160
EGD38300.1

F0IUP5


Streptococcus sanguinis SK330
EGF15598.1

F2C6E1


Streptococcus sanguinis SK340
EGJ44393.1

F3UHS5



EGQ20720.1

F9E0L5



EGQ24079.1

F9E949


Streptococcus sanguinis SK353
EFX93874.1
ZP_08087316.1
E8KPS1



EGC23179.1

F0FDK9


Streptococcus sanguinis SK355
EGJ38546.1

F3US14


Streptococcus sanguinis SK408
EGC24921.1

F0FJT3



EGF08624.1

F2BIW4



EGF19580.1

F2CDE0


Streptococcus sanguinis SK49
EGJ38526.1

F3UY06


Streptococcus sanguinis SK678
EGC27602.1

F0FR11


Streptococcus sanguinis SK72
EGD29398.1

F0I1T9


Streptococcus sp. SK140
EIF38589.1
ZP_10038759.1


Streptococcus sp. SK643
EIF39120.1
ZP_10040082.1


Streptococcus sp. C300
EFX56812.1
ZP_08050022.1
E9FHQ0


Streptococcus sp. M143
EFA24288.1
ZP_06198674.1
D2EQA3


Streptococcus sp. M334
EFX59032.1
ZP_08050711.1
E9FKM3


Streptococcus sp. oral taxon 056 str.
EGP66993.1
ZP_08662553.1
F9HGU8


F0418


Streptococcus sp. oral taxon 058 str.
EHI77041.1
ZP_09174472.1
G6C955


F0407


Streptococcus sp. oral taxon 071 str.
EFM35756.1
ZP_07458299.1
E0Q0E1


73H25AP


Streptococcus thermophilus (strain
ABJ66404.1
YP_006340266.1
Q03K68


ATCC BAA-491/LMD-9)
AFJ83601.1
YP_820600.1


Streptococcus urinalis 2285-97
EHJ57402.1
ZP_09137413.1
G5KGX7


Streptococcus vestibularis ATCC 49124
EFQ59772.1
ZP_07722949.1
E3CNS3



EFX96303.1
ZP_08069523.1
E8KUL8


Tannerella forsythia (strain ATCC
AEW20074.1
YP_005013402.1
G8UKW3


43037/JCM 10827/FDC 338) Bacteroides


forsythus


Tepidanaerobacter acetatoxydans
AEE92466.1
YP_004461773.1
F4LT66


(strain DSM21804/JCM 16047/RE1)


Thermincola potens (strain JR)
ADG83236.1
YP_003641137.1
D5XAN2


Thermovirga lienii (strain ATCC BAA-
AER67504.1
YP_004933601.1
G7V8P3


1197/DSM 17291/Cas60314)


Treponema sp. JC4
EID85541.1
ZP_10009441.1
I0XA75


Turicibacter sanguinis PC909
EFF63439.1
ZP_06622212.1
D4W6H2


Turicibacter sp. HGF1
EGC92139.1
ZP_08167528.1
F0HEZ8


Victivallis vadensis ATCC BAA-548
EFA99410.1
ZP_06244492.1
D1N9V2


Weeksella virosa (strain ATCC
ADX67941.1
YP_004238519.1
F0P329


43766/DSM 16922/JCM 21250/NBRC


16016/NCTC 11634/CL 345/78)


Weissella confusa LBAE C39-2
CCF29905.1
ZP_10257557.1
H1X5Q2


Weissella paramesenteroides ATCC
EER75253.1
ZP_04782492.1
C5R921


33313


Weissella thailandensis fsh4-2
CCC57018.1

G0UGX7










Eukaryota










Organism
GenBank ID
RefSeq
UniProt





Arabidopsis lyrata subsp. lyrata (Lyre-
EFH68720.1
XP_002892461.1
D7KIS7


leaved rock-cress)


Arabidopsis lyrata subsp. lyrata (Lyre-
EFH61940.1
XP_002885681.1
D7L5U9


leaved rock-cress)


Arabidopsis thaliana (mouse-ear cress)
CAA85388.1

Q42562


Arabidopsis thaliana (mouse-ear cress)
CAA85387.1

Q42558


Arabidopsis thaliana (mouse-ear cress)
AEE76980.1
NP_189147.1
Q42561



BAB02069.1



BAD43868.1



CAA85389.1


Arachis hypogaea (peanut)
ABO38556.1

A9P5P3



ABO38557.1


Arachis hypogaea (peanut)
ABO38558.1

A9P5P5


Arachis hypogaea (peanut)
ABO38554.1

A9P5P1


Arachis hypogaea (peanut)
ABO38555.1

A9P5P2


Brachypodium sylvaticum (false brome)
ABL85052.1

A1YKG2


Brassica campestris (field mustard)
AAC49002.1

Q39402


Brassica juncea (Indian mustard)
CAC14164.1

Q9FT16


(Sinapis juncea)


Camellia oleifera
ACQ57189.1

C3W2Q2


Camellia oleifera
ACQ57190.1

C3W2Q3


Camellia oleifera
ACQ63293.1

C3W2Q1


Camellia oleifera
ACQ57188.1

C3W2Q0


Camellia oleifera
ACQ57187.1

C3W2P9


Capsicum annuum (bell pepper)
ADH03021.1

D7RU32


Capsicum chinense (Scotch bonnet)
AAG35064.1

Q9FPM5


(bonnet pepper)


Capsicum frutescens (cayenne papper)
AEO12091.1

G3F052


(Tabasco pepper)


Chimonanthus praecox
ADM18137.1

H9L9E6


Chlamydomonas reinhardtii
EDP08596.1
XP_001696619.1
A8HY17


(Chlamydomonas smithii)


Citrus sinensis (sweet orange) (Citrus
AEX99667.1

H2ESB6


aurantium var. sinensis)


Coccomyxa subellipsoidea C-169
EIE27817.1

I0ZAZ8


Cocos nucifera (coconut)
AEM72519.1

G3ESU6


Cocos nucifera (coconut)
AEM72520.1

G3ESU7


Cocous nucifera (coconut)
AEM72521.1

G3ESU8


Cuphea calophylla subsp. mesostemon
ABB71581.1

Q2A1N7


Cuphea calophylla subsp. mesostemon
ABB71579.1

Q2Q1N8



ABB71580.1


Cuphea hookeriana (cigar plant)
AAC72882.1

Q9ZTF8


Cuphea hookeriana (cigar plant)
AAC72881.1

Q9ZTF9


Cuphea hookeriana (cigar plant)
AAC49269.1

Q39514


Cuphea hookeriana (cigar plant)
AAC72883.1

Q9ZTF7


Cuphea lanceolata (cigar flower)
CAA54060.1

Q39534


Cuphea lanceolata (cigar flower)
CAC19933.1

Q9FNS8


Cuphea lanceolata (cigar flower)
CAC19934.1

Q9FNS7


Cuphea lanceolata (cigar flower)
CAB60830.1

Q9SMI9


Cuphea palustris
AAC49180.1

Q39555


Cuphea palustris
AAC49179.1

Q39554


Cuphea viscosissima
AEM72523.1

G3ESV0


Cuphea viscosissima
AEM72524.1

G3ESV1


Cuphea viscosissima
AEM72522.1

G3ESU9


Cuphea wrightii (Wright's waxweed)
AAC49784.1

Q39663


Cuphea wrightii (Wright's waxweed)
AAC49783.1

Q39662


Garcinia mangostana
AAB51525.1

O04794


Garcinia mangostana
AAB51524.1

O04793


Garcinia mangostana
AAB51523.1

O04792


Glycine max (soybean) (Glycine hispida)
ABD91726.1
NP_001237802.1
B1MVG6


Glycine max (soybean) (Glycine hispida)
ABI20759.1

Q0GJK0


Glycine max (soybean) (Glycine hispida)
ABI20760.1

Q0GJJ9


Haematococcus pluvialis
AEF13160.1

G9B653


Helianthus annuus (common sunflower)
CAC80371.1

Q8VXJ5


Helianthus annuus (common sunflower)
AAB88824.1

O48568



AAX19377.1



AAX19378.1



AAX19379.1



AAX19380.1



AAX19381.1



AAX19382.1



AAX19383.1



AAX19384.1



AAX19385.1



AAX19386.1



AAX19387.1


Helianthus annuus (common sunflower)
AAQ08202.1

Q2UZT2


Helianthus annuus (common sunflower)
CAC80370.1

Q8VXJ6


Helianthus annuus (common sunflower)
AAQ08223.1

Q2UZT0



AAQ08224.1



AAQ08225.1



AAQ08226.1


Helianthus annuus (common sunflower)
AAL79361.1

Q6K1M5


Helianthus annuus (common sunflower)
AAX54515.1

Q4KU00


Helianthus annuus (common sunflower)
AAX54514.1

Q4KU01


Helianthus annuus (common sunflower)
AAX54516.1

Q4KTZ8



AAX54517.1



AAX54518.1



AAX54519.1



AAX54520.1



AAX54521.1



AAX54522.1



AAX54523.1



AAX54524.1



AAX54525.1



AAX54526.1



AAX54527.1


Iris germanica (flag) (fleur-de-lis)
AAG43857.1

Q9FQY1


Iris germanica (flag) (fleur-de-lis)
AAG43858.1

Q9FQY0


Iris germanica (flag) (fleur-de-lis)
AAG43859.1

Q9FQX9


Iris tectorum
AAG43860.1

Q9FQX8


Iris tectorum
AAG43861.1

Q9FQX7


Iris tectorum
AAL77443.1

Q8S9G4


Jatropha curcas
ABX82799.3

A9UFC5


Macadamia tetraphylla
ADA79524.1

D2E6T0


Macadamia tetraphylla
ACB29661.1

D5FFZ2


Medicago truncatula (barrel medic)
AES91389.1
XP_003609192.1
G7JU47


(Medicago tribuloides)


Myristica fragrans (nutmeg)
AAB71729.1

O24419


Myristica fragrans (nutmeg)
AAB71730.1

Q6I8R5


Nicotiana tabacum (common tobacco)
AFE88232.1

H9CCH9


Perilla frutescens (beefsteak mint)
AAL77445.1

Q8SMI2


(Perilla ocymoides)


Populus tomentosa (Chinese white
ABC47311.1

A1XAM4


poplar)


Triticum aestivum (wheat)
CAD32683.1

Q8L6B1


Ulmus americana (American elm)
AAB71731.1

O24420


Umbellularia californica (California bay
AAC49001.1

Q41634


laurel) (Tetranthera californica)





information obtained from www.enzyme.cbirc.iastate.edu (© Iowa State University of Science and Technology; used with permission)






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 FIG. 1, starting at the amino acid position indicated by ▾). 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). “Corresponding to” is used herein to refer to an amino acid in an acyl-ACP TE, such as a plant acyl-ACP TE, which, when the amino acid sequence of the acyl-ACP TE is aligned with the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by FatB2 (CvFatB2), aligns with the position of the amino acid of the Cuphea viscosissima acyl-ACP TE to which reference is made by number. The amino acid in the acyl-ACP TE may or may not have the same numbered position as that in 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). Mutating amino acid 133 to phenylalanine (F) or leucine (L) can increase production of C8 fatty acids, mutating amino acid 133 to valine (V) or alanine (A) can increase production of C14/16 fatty acids, mutating amino acid 139 to isoleucine (I) can increase production of C8-C12 fatty acids (e.g., C8 fatty acids), mutating amino acid 139 to asparagine (N) can increase production of C14/16 fatty acids, mutating amino acid 142 to A and mutating amino acid 143 to serine (S) can increase production of C8 fatty acids, and mutating both of amino acids 142 and 143 to arginine (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. 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). 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. Generally speaking, such mutations alter the level of activity of the plant acyl-ACP TE, such as increase the level of activity of the plant acyl-ACP TE, without altering the substrate specificity significantly, if at all. In other words, these mutations can alter the total amount of fatty acids produced, such as increase the total amount of fatty acids produced, but do not significantly alter (if at all) the mole percentage of each fatty acid. Preferably, even desirably, the level of activity of the plant acyl-ACP TE is increased, rather than decreased.
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 FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (see, e.g., family TE14; which website is incorporated herein by reference for its teachings regarding acyl-ACP TEs), in particular an acyl-ACP TE from a bacterium, e.g., an acyl-ACP TE from Bryantella formatexigens, is also provided. The mutant acyl-ACP TE 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.
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., FIG. 5, FIG. 9, Table 1, Table 2, and www.enzyme.cbirc.iastate.edu (see, e.g., family TE14), which website is hereby incorporated by reference for its teachings regarding acyl-ACP TEs). Preferably, the acyl-ACP TE gene is another Cuphea viscosissima acyl-ACP TE gene. The chimeric Cuphea viscosissima acyl-ACP TE gene preferably is a chimeric FatB1 gene or a chimeric FatB2 gene. In this regard, preferably the chimeric CvFatB1 gene comprises a segment of the CvFatB2 gene or the chimeric CvFatB2 gene comprises a segment of the CvFatB2 gene.
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., FIG. 5, FIG. 9, Table 1, Table 2, and www.enzyme.cbirc.iastate.edu (see, e.g., family TE14), which website is hereby incorporated by reference for its teachings regarding acyl-ACP TEs). 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 (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., FIG. 5, FIG. 9, Table 1, Table 2, and www.enzyme.cbirc.iastate.edu (see, e.g., family TE14, which website is hereby incorporated by reference for its teachings regarding acyl-ACP TEs). The isolated or purified nucleic acid molecule can be a vector. The encoded mutant plant acyl-ACP TE can comprise 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 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).
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 FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (see, e.g., family TE14; which website is incorporated by reference herein for its teachings regarding acyl-ACP TEs), in particular an acyl-ACP TE from a bacterium, e.g., an acyl-ACP TE from Bryantella formatexigens (nucleotide sequence is SEQ ID NO: 23; amino acid sequence is SEQ ID NO: 24), is also provided. The mutant acyl-ACP TE 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. Preferably, the mutant acyl-ACP TE derived from wild-type Bryantella formatexigens acyl-ACP TE has altered substrate specificity such that it increases production of short-chain fatty acids. Depending on the particular acyl-ACP TE (e.g., an acyl-ACP TE from FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (see, e.g., family TE14; which website is incorporated by reference herein for its teachings regarding acyl-ACP TEs), such as acyl-ACP TE from a bacterium) that has been mutated, such mutants can have altered substrate specificity, such as altered substrate specificity that results in increased production of short-chain fatty acids or fatty acids of longer chain length, and/or altered level of activity, e.g., thioesterase activity, such as the total amount of fatty acids produced. Mutants can be derived from wild-type acyl-ACP TEs in accordance with methods known in the art (e.g., site-directed mutagenesis) and exemplified herein. Due to the degeneracy of the genetic code, the same amino acid sequence can be encoded by nucleotide sequences that vary quite a bit.
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 FIG. 5, FIG. 9, Table 1, Table 2, or www.enzyme.cbirc.iastate.edu (see, e.g., family TE14; which website is incorporated by reference herein for its teachings regarding acyl-ACP TEs). Preferably, the acyl-ACP TE is another Cuphea viscosissima acyl-ACP TE. In this regard, the chimera can be made in accordance with methods known in the art, such as at the level of DNA or protein, including synthesis. Preferably, the chimera is a chimera of the TE encoded by a FatB1 gene or a chimera of the TE encoded by a FatB2 gene. In this regard, the chimera can be a chimera of the TE encoded by the CvFatB1 gene comprising a segment of the TE encoded by the CvFatB2 gene or a chimera of the TE encoded by the CvFatB2 gene comprising a segment of the TE encoded by the CvFatB1 gene.
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.
EXAMPLES
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.
Example 1
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 (www.enzyme.cbirc.iastate.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:




  z
  =
  
    
      
        
          x
          _
        
        ij
      
      -
      
        
          (
          
            
              
                x
                _
              
              ii
            
            +
            
              
                x
                _
              
              jj
            
          
          )
        
        ⁢
        
          /
        
        ⁢
        2
      
    
    
      
        
          
            σ
            i
            2
          
          
            n
            ij
          
        
        +
        
          
            σ
            ii
            2
          
          
            n
            ii
          
        
        +
        
          
            σ
            jj
            2
          
          
            n
            jj
          
        
      
    
  




where xij, xii, and xjj are the inter- and intra-subfamily mean JTT distances, nij, nii, and njj are the total number of taxa used for each x value, and σi2, σii2, and σjj2 are the pooled inter- and intra-subfamily variances (Mertz et al., Biopolymers 79: 197-206 (2005)).

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).







TABLE 2







Total activity of synthesized and cloned acyl-ACP TEs















Total Fatty



ACC No.


Acids


Subfamily
or Name
Organism
Rationale for synthesisa
(nmol/mL)b










Kingdom: Planta











A
AAC49179c,d
Cuphea palustris
A (Bimodal specificity for C8 and C10
708 ± 45





substrates)





Dehesh et al., Plant Physiol. 110: 203-210





(1996)



AAB71731
Ulmus
A (Broad specificity; highest activity on
1098 ± 62 




Americana
C10 and C16)





Voelker et al., Plant Physiol. 114: 669-677





(1997)



AAG43857
Iris germanica
B
261 ± 20



AAG43858
Iris germanica
B
14.8 ± 4.6



EER87824
Sorghum bicolor
B (Member of a Subfamily A Poeceae TE
126 ± 13





cluster)



EER88593
Sorghum bicolor
B (Member of a Subfamily A Poeceae TE
90.7 ± 8.0





cluster)



CnFatB1
Cocos nucifera
C
130 ± 12



CnFatB2
Cocos nucifera
C
572 ± 32



CnFatB3
Cocos nucifera
C
200 ± 11



CvFatB1
Cuphea
C
79.2 ± 9.7




viscosissima



CvFatB2
Cuphea
C
249 ± 9 




viscosissima



CvFatB3
Cuphea
C
18.9 ± 2.1




viscosissima



AAD42220
Elaeis
C
36.7 ± 3.8




guineensis


B
EDQ65090
Physcomitrella
B (Member of novel plant subfamily)
380 ± 29




patens



EER96252
Sorghum bicolor
B (Member of novel plant subfamily)
175 ± 11



EES11622
Sorghum bicolor
B (Member of novel plant subfamily)
 9.43 ± 2.03


D
EEH52851
Micromonas
B
16.3 ± 1.6




pusilla







Kingdom: Bacteria











E
ACL08376
Desulfovibrio
D (Medium-chain linear, branched, and
330 ± 9 




vulgaris
hydroxy fatty acids)





Ratledge et al., Microbial Lipids, Vol. 1,





Academic Press, San Diego, CA (1988)


F
CAH09236
Bacteroides
D (Hydroxy fatty acids)
215 ± 6 




fragilis
Ratledge et al., Microbial Lipids, Vol. 1,





Academic Press, San Diego, CA (1988)



ABR43801
Parabacteroides
D (Branched and branched hydroxy fatty
70.3 ± 4.4




distasonis
acids)





Sakamoto et al., Int. J. Syst. Evol.





Microbiol. 57: 293-296 (2007)



AAO77182e
Bacteroides
D (Anteiso-branched and hydroxy fatty
60.4 ± 2.9




thetaiotaomicron
acids)





Ratledge et al., Microbial Lipids, Vol. 1,





Academic Press, San Diego, CA (1988)


G
ABG82470
Clostridium
D (Medium-chain fatty acids)
72.0 ± 9.5




perfringens
Moss et al., Appl. Microbiol. 15: 390-397





(1967)


H
EEG55387
Clostridium
B
25.9 ± 4.2




asparagiforme



EET61113
Bryantella
B
381 ± 3 




formatexigens


I
EDV77528
Geobacillus sp.
D (Iso-branched fatty acids)
 64.9 ± 12.0





Rahman et al., BMC Microbiol. 2007: 7





(2007)


J
BAH81730
Streptococcus
D (Medium-chain and cyclic propane ring
623 ± 14




dysgalactiae
fatty acids) Ratledge et al., Microbial Lipids,





Vol. 1, Academic Press, San Diego, CA





(1988)



ABJ63754
Lactobacillus
D (Medium-chain and cyclic propane ring
710 ± 10




brevis
fatty acids) Johnsson et al., Appl. Environ.





Microb. 61: 4497-4499 (1995)



CAD63310e
Lactobacillus
D (Medium-chain 3′-hydroxy fatty acids)
436 ± 10




plantarum
Johnsson et al., Appl. Environ. Microb. 61:





4497-4499 (1995); Sjogren et al., Appl.





Environ. Microb. 69: 7554-7557 (2003)


Non-
EEI82564
Anaerococcus
D (Organism produces butyric acid)
1381 ± 146


grouped

tetradius
Murdoch et al., J. Med. Microbiol. 34: 295-308





(1991)



CAE80300
Bdellovibrio
D (Straight-chain odd-numbered fatty acids)
333 ± 18




bacteriovorus
Ratledge et al., Microbial Lipids, Vol. 1,





Academic Press, San Diego, CA (1988)



ABN54268
Clostridium
D (Branched-chain fatty acids)
97.7 ± 3.2




thermocellum
Ratledge et al., Microbial Lipids, Vol. 1,





Academic Press, San Diego, CA (1988)





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. TEs were expressed in E. coli K27, and free fatty acids (FAs) that accumulated in the medium were analyzed by GC-MS.






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.
Example 2
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 SuperScriptTM 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.
Example 3
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 FIG. 2. Acyl-ACP TEs from C. palustris and U. americana, which have previously been functionally characterized in vitro (Dehesh et al. (1996), supra; and Voelker et al. (1997), supra), were studied as controls.
C. palustris acyl-ACP TE produced 97 mol % 8:0 and only 0.8 mol % 10:0 fatty acids (FIG. 2A), while U. americana acyl-ACP TE made 44 mol % 8:0 and 23 mol % 10:0 fatty acids (FIG. 2B). E. guineensis acyl-ACP TE produced mainly 14:0 (47 mol %) and 16:1 (26 mol %) fatty acids (FIG. 2C). The acyl-ACP TEs from I. germanica and S. bicolor have similar substrate specificities, producing mainly 14:0 (30-46 mol %), 16:0 (11-23 mol %), and 16:1 (31-44 mol %) fatty acids (FIG. 2B). CnFatB1 (JF338903) and CnFatB2 (JF338904) made predominantly 14:0 (36-44 mol %) and 16:1 (31-44 mol %) fatty acids, whereas CnFatB3 (JF338905) made mainly 12:0 (34 mol %) and 14:1 (22 mol %) fatty acids (FIG. 2C). Finally, CvFatB1 (JF338906) produced mainly 8:0 (51 mol %) and 10:0 (25 mol %), and CvFatB2 (JF338907) made mainly 14:0 (46 mol %), 16:0 (25 mol %) and 16:1 (20 mol %) fatty acids (FIG. 2A). In contrast, CvFatB3 (JF338908) has narrower substrate specificity, producing predominantly 14:0 fatty acid (84 mol %).
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 (FIG. 2D).
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 FIG. 3). Based on their substrate specificities, these acyl-ACP TEs were classified into two groups. One group produced primarily SCFAs and MCFAs (>75 mol % 4:0 to 8:0 fatty acids). This group included acyl-ACP TEs from Anaerococcus tetradius (GenBank:EE182564, no subfamily, 87% 8:0), Clostridium perfringens (GenBank:ABG82470, Subfamily G, 14% 6:0 and 70% 8:0), Lactobacillus brevis (GenBank:ABJ63754, Subfamily J, 7% 4:0, 14% 6:0, and 55% 8:0), and Lactobacillus plantarum (GenBank:CAD63310, Subfamily J, 11% 6:0 and 68% 8:0) (FIG. 3). The other group showed broad- and binary-range substrate specificities. The binary-range activities were centered on C8 and C12/C14 substrates (FIG. 3).
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 FIG. 4).
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.
Example 4
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 (FIG. 5). Class I consists of thirteen plant acyl-ACP TEs from Subfamilies A, B, and D. Class II contains eleven acyl-ACP TEs, ten from bacteria in Subfamilies E, F, H, I, and J, and a non-grouped sequence, and only one from a plant (CnFatB3) in Subfamily A. Class III includes seven acyl-ACP TEs, of which three are from plants in Subfamily A and four are from bacteria in Subfamilies G and J and a non-grouped sequence. Considering the previously characterized class of oleoyl-ACP TEs in Subfamily C, TE14 members may now be sorted into four classes based on their substrate specificities.
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).
Example 5
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.
Example 6
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.
Example 7
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 FIG. 2. The data of all mutants (blue bars) were from single analysis, while the activity of EET61113 (red bar) was presented as the average of 16 replicates. A total of 27 mutants showed higher acyl-ACP TE activity than wild-type, among which 8 mutants showed more than 20% increase and 2 mutants (TE20MT156 and TE20MT180) had  ̃40% increase. While activity increased slightly in the site-directed mutants TE20-N169Y and TE20-S222I as compared to wild-type EET61113, activity increased dramatically in a random mutant (designated TE20MT9) containing the N169Y and S222I mutations. Thus, surprisingly the double mutation had a synergistic effect, as opposed to an additive effect, on activity by the mutant acyl-ACP TE. While the wild-type TE produced approximately 50 nmol/ml culture of 4:0 fatty acids and approximately 110 nmol/ml culture of 6:0 fatty acids, the mutant designated TE20MT9 produced approximately 45 nmol/ml culture of 4:0 fatty acids and approximately 230 nmol/ml culture of 6:0 fatty acids.
Example 8
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 FIG. 7).







TABLE 3







Primers for amplification of the fragments I through VI









Fragments
Forward primer
Reverse primer





I
pUC57R (5′-CGGCTCGTATGTTGTGTGGAAT-
Cv-R2 (5′-GGTACGATCCGCGCCGATTTC-3′)



3′) [SEQ ID NO: 12]
[SEQ ID NO: 18]


 


II
Cv-F2 (5′-GAAATCGGCGCGGATCGTACC-3′)
Cv-R3 (5′-CCAGGTCGGATAACGATTGAC-3′)



[SEQ ID NO: 13]
[SEQ ID NO: 19]


 


III
Cv-F3 (5′-GTCAATCGTTATCCGACCTGG-3′)
Cv-R4 (5′-CGGGTTTTCTGGTTCATCAT-3′)



[SEQ ID NO: 14]
[SEQ ID NO: 20]


 


IV
Cv-F4 (5′-ATGATGAACCAGAAAACCCG-3′)
Cv-R5 (5′-TCGTTCCAACGCGGCGTCAGACC-3′)



[SEQ ID NO: 15]
[SEQ ID NO: 21]


 


V
Cv-F5 (5′-GGTCTGACGCCGCGTTGGAACGA-3′)
Cv-R6 (5′-ATCTTCCAGACGCAGCAG-3′)



[SEQ ID NO: 16]
[SEQ ID NO: 22]


 


VI
Cv-F6 (5′-CTGCTGCGTCTGGAAGAT-3′)
pUC57F (5′-CTGCAAGGCGATTAAGTTGGGTAAC-3′)



[SEQ ID NO: 17]
[SEQ ID NO: 11]









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.
Example 9
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 FIG. 17). Multiple sequence alignment and phylogenetic analysis resulted in the identification of another nine residues that also may affect substrate specificities, namely residue 110 in Fragment II, residues 133 and 139 in Fragment III, and residues 173, 176, 184, 192, 198, and 205 in Fragment IV (see FIG. 9, which lists the amino acid present at a given position for the indicated organism). The presence of leucine (L) or valine (V) at position 110, phenylalanine (F) or leucine (L) at position 133, isoleucine (I) at position 139, phenylalanine (F) or leucine (L) at position 173, leucine (L) at position 176, isoleucine (I), phenylalanine (F) or leucine (L) at position 184, proline (P) at position 192, asparagine (N) or serine (S) at position 198, and phenylalanine (F) at position 205 were associated with the production of mainly C8 fatty acids. The production of mainly C14/16 fatty acids was associated with the presence of valine (V) or leucine (L) at position 110, valine (V) or alanine (A) at position 133, asparagine (N) at position 139, leucine (L) at position 173, isoleucine (I), phenylalanine (F), valine (V), or methionine (M) at position 176, isoleucine (I; or, in one instance, leucine (L)) at position 184, threonine (T), serine (S), valine (V), alanine (A), proline (P), or aspartic acid (D) at position 192, aspartic acid (D) at position 198, and leucine (L) at position 205. Site-directed mutagenesis and fatty acid profiling were used for verification.
Example 10
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 FIG. 10, which shows the fatty acid profiles of the mutants on the right (wherein the dark shading highlights the major fatty acids produced by the TEs), the cluster analysis of the fatty acid profiles on the left, and the point mutations in each mutant in the middle. The wild-type enzyme is CvFatB2, which is shown with light stippling. Heavy stippling indicates residues that have been mutated to the amino acid present at the corresponding position in CvFatB1. Cross-hatching indicates residues that have been mutated to amino acids that are not present in CvFatB1 or CvFatB2.
Example 11
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 FIGS. 8a and 8b. As shown in FIGS. 8a and 8b, the biggest variances among the chimeric TEs were observed for 8:0 and 14:0 fatty acids. The mole percentages of 8:0 and 14:0 fatty acids for all chimeric TEs were plotted, and it was determined that the replacement of Fragment III changed the fatty acid profile most dramatically. Thus, Fragment III was determined to contain the most important residues for substrate specificity, although Fragments I, II, and IV also affected substrate specificity.
Example 12
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 (www.ebi.ac.uk/Tools/msa/clustalw2/) (see FIG. 11). The N-terminal chloroplastic transit peptide and the proposed hydrophobic membrane anchor (Facciotti et al., Fett-Lipid 100(4-5): 167-172 (1998)), which can be experimentally removed without affecting TE activity and specificity of acyl-ACP TEs, were not included. Conserved and variable residues were identified. Among the 307 residues, 65 were chosen for random mutagenesis with 2-8 possible substitutions at each position, and consensus sequences were used for the other positions. The protein sequence was back-translated into nucleotide sequence with the GeneDesign algorithm (54.235.254.95/gd/) (Richardson et al., Genome Research 16(4): 550-556 (2006)). A total of 30 DNA oligos were designed to assemble the TE mutant library, with mixed nucleotides incorporated into the corresponding positions for variable residues. There was an overlap of 22-25 nucleotides between oligos with Tm values of 54-56° C. Restriction sites of Bam HI and Eco RI were incorporated into the first and last oligos. The theoretical maximum number of variants in the library was 1038.
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 (FIG. 12). The pUCHisGm vector was modified from pUC57 by the insertion of a 6×His tag at the N-terminus of the cloned TE gene and the fusion of a gentamicin-resistant gene (GmR) at the C-terminus of the cloned TE gene. When cloned into the pUCHisGm vector, the acyl-ACP TE variant gene was fused to the GmR gene with a 3×GGGS [SEQ ID NO: 35] flexible linker (Chen et al., Adv. Drug. Deliv. Rev. 65: 1357-1369 (2013)) between them and transcriptionally controlled by a weak lacZ promoter. The constructed vectors containing the variant genes were transformed in E. coli K27 by electroporation. E. coli K27 carries a mutation in the acyl-CoA synthetase gene (fadD) and is thus capable of accumulating free fatty acids. The six wild-type acyl-ACP TEs (UaFatB1, CpFatB1, CvFatB1, CvFatB2, CnFatB2, and CnFatB3) were also cloned into pUCHisGm and transformed into E. coli K27 as controls.
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 FIG. 14, which is a bar graph showing the fatty acid production of parental TEs (the first five bars and the 40th bar) and TE variants. Bars represent the total fatty acids in μM, and dots represent the total fatty acids in mg/L.
Example 13
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 FIG. 14; SEQ ID NO: 31). FIG. 13 is a sequence alignment of the TE variant TEGm162 (SEQ ID NO: 31) with mature CvFatB2 (SEQ ID NO: 32). This sequence was not discovered in 47 randomly sequenced variants isolated from the non-screening plate, suggesting that the Neutral Red plate strongly screened for this particular variant. Noteworthy are the following differences (based on the mature CvFatB2 sequence): amino acids 23-25, 27, 32, 36, 38, 47, 74, 78, 81-82, 87-88, 91, 110, 115, 17-118, 133-134, 142, 148, 151, 155-157, 160, 178, 184-185, 187, 189, 193, 195, 198, 203, 205, 207, 210, 214-215, 224, 236, 239, 243, 245, 251, 253-254, 256-257, 267, 275-277, 280-281, 283-285, 287-288, 297, 313, and 316-325. With 147 analyses, the average fatty acid production of TEGm162 was 1,173±207 μM (mean±SD), which is about 26% higher than the productivity of CpFatB1 and ten-fold higher than the productivity of CnFatB3. Among the other sequenced variants, another three had the identical sequence, referred to herein as TEGm204. Including TEGm162 and TEGm204, 27 distinct sequences were identified from 177 sequenced acyl-ACP TE variants. Among them, three variants had N-terminal truncations, and one variant had a C-terminal truncation; they still produced higher levels of fatty acids. The variant with the C-terminal truncation lacked 138 amino acids and was not analyzed further. The protein sequences of 26 distinct variants and the six parental TEs were subjected to phylogenetic analysis. The phylogenetic tree, which is shown in FIG. 15a, indicated that the closest sequence to the six parental TEs was TEGm419, which had 78% sequence identity with CpFatB1 but showed broad substrate specificity. Some TE variants had similar sequences but displayed different substrate specificities, which can be used to identify residues that determine specificity of acyl-ACP TE.
The fatty acid profiles of the 26 distinct variants and the six parental TEs were evaluated (see FIG. 15b). Various substrate specificities were exhibited. Sixteen variants produced mainly 14/16-carbon fatty acids; four variants produced mainly 8-carbon fatty acid; and six variants produced evenly distributed 8- to 16-carbon fatty acids. TEGm205 and TEGm258 had very similar protein sequences but different substrate specificities (see FIG. 15b). By comparing the sequences of TEGm205 [SEQ ID NO: 34] and TEGm258 [SEQ ID NO: 33], nine amino acid residues in the N-terminal one-third of the enzyme differed between the two variants (see FIG. 16). Among these, residue 92 (numbered based on sequence of TEGm258) has been shown to affect substrate specificity. Another three residues are in the region 55-67 (amino acids being numbered according to CvFatB2 mutant lacking 18 amino acids; this region corresponds to amino acids 73-85 of mature CvFatB2 and amino acids 157-169 of CvFatB2 with N-terminal transit peptide), which is implicated in substrate specificity. Comparison of TEGm202 and TEGm157, as well as TEGm201 and TEGm245, led to the identification of other residues in this region.
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 S222I 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.

## Claims

1. 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, the amino acid sequence of which is at least 85% identical to the amino acid sequence of SEQ ID NO:3, 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 SEQ ID NO:3, alone or in further combination with a mutation of at least one amino acid in the region corresponding to amino acids 73-85 of SEQ ID NO:3,
wherein SEQ ID NO:3 is the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by the FatB2 gene,
whereupon the specificity of the plant acyl-ACP TE for at least one of its substrates is altered.

2. The method of claim 1, 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 SEQ ID NO:3.

3. The method of claim 1, 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 SEQ ID NO:3, whereupon the level of activity of the plant acyl-ACP TE is altered.

4. The method of claim 2, 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 SEQ ID NO:3, whereupon the level of activity of the plant acyl-ACP TE is altered.

5. 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, which 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 SEQ ID NO:3, and
(ii) introducing into the plant acyl-ACP TE, the amino acid sequence of which is at least 85% identical to the amino acid sequence of SEQ ID NO:3, 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 SEQ ID NO:3, alone or in further combination with a mutation of at least one amino acid in the region corresponding to amino acids 73-85 of SEQ ID NO:3,
wherein SEQ ID NO:3 is the mature amino acid sequence of the Cuphea viscosissima acyl-ACP TE encoded by the FatB2 gene,
whereupon the level of activity of the plant acyl-ACP TE and the specificity of the plant acyl-ACP TE for at least one of its substrates are altered.

6. The method of claim 5, 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 SEQ ID NO:3.