# Patent

## Title
Thioesterases and cells for production of tailored oils

## Abstract

The invention features plant acyl-ACP thioesterase genes of the FatB class and proteins encoded by these genes. The genes are useful for constructing recombinant host cells having altered fatty acid profiles. Oleaginous microalga host cells with the new genes or previously identified FatB genes are disclosed. The microalgae cells produce triglycerides with useful fatty acid profiles.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 3, 2013, is named SOLAP019US_SL.txt and is 318,250 bytes in size.
BACKGROUND
Certain organisms including plants and some microalgae use a type II fatty acid biosynthetic pathway, characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis. In contrast, mammals and fungi use a single, large, multifunctional protein.
Type II fatty acid biosynthesis typically involves extension of a growing acyl-ACP (acyl-carrier protein) chain by two carbon units followed by cleavage by an acyl-ACP thioesterase. In plants, two main classes of acyl-ACP thioesterases have been identified: (i) those encoded by genes of the FatA class, which tend to hydrolyze oleoyl-ACP into oleate (an 18:1 fatty acid) and ACP, and (ii) those encoded by genes of the FatB class, which liberate C8-C16 fatty acids from corresponding acyl-ACP molecules.
Different FatB genes from various plants have specificities for different acyl chain lengths. As a result, different gene products will produce different fatty acid profiles in plant seeds. See, U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and 5,344,771; 5,304,481. Recently, FatB genes have been cloned into oleaginous microalgae to produce triglycerides with altered fatty acid profiles. See, WO2010/063032, WO2011,150411, and WO2012/106560.
SUMMARY
In an embodiment of the invention, there is a nucleic acid having at least 80% sequence identity to any of SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76 or any equivalent sequences by virtue of the degeneracy of the genetic code.
In another embodiment of the invention, there is a nucleic acid sequence encoding a protein having at least 80% sequence identity to any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, or 77, or a fragment thereof having acyl-ACP thioesterase activity. The protein can have acyl-ACP thioesterase activity operable to alter the fatty acid profile of an oil produced by a recombinant cell comprising that sequence.
In a further embodiment of the invention there is a method of producing a recombinant host cell that produces an altered fatty acid profile, the method comprising transforming the cell with any of the nucleic acids mentioned above. The host cell can be a plant cell, a microbial cell, or a microalgal cell. Another embodiment of the invention includes a host cell produced by this method.
In an embodiment, there is a method for producing an oil or oil-derived product, the method comprising cultivating the host cell and extracting the oil, optionally wherein the cultivation is heterotrophic growth on sugar. Optionally, a fatty acid, fuel, chemical, or other oil-derived product can be produced from the oil. Optionally, the oil can have a fatty acid profile comprising at least 20% C8, C10, C12, C14 or C16 fatty acids. Optionally, the oil is produced by a microalgae and can lack C24-alpha sterols.


DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION
Definitions
As used with respect to nucleic acids, the term “isolated” refers to a nucleic acid that is free of at least one other component that is typically present with the naturally occurring nucleic acid. Thus, a naturally occurring nucleic acid is isolated if it has been purified away from at least one other component that occurs naturally with the nucleic acid.
A “natural oil” or “natural fat” shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the natural oil or natural fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. In connection with a natural oil or natural fat, and as used generally throughout the present disclosure, the terms oil and fat are used interchangeably, except where otherwise noted. Thus, an “oil” or a “fat” can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term “fractionation” means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms “natural oil” and “natural fat” encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching and/or degumming, that does not substantially change its triglyceride profile. A natural oil can also be a “noninteresterified natural oil”, which means that the natural oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.
“Exogenous gene” shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a “transgene”. A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
“Fatty acids” shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.
“Microalgae” are microbial organisms that contain a chloroplast or other plastid, and optionally that are capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca. 
An “oleaginous” cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An “oleaginous microbe” or “oleaginous microorganism” is a microbe, including a microalga that is oleaginous.
In connection with a natural oil, a “profile” is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A “fatty acid profile” is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID). The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids.
“Recombinant” is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode a gene product or suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
Thioesterase Sequences
Additional FatB genes encoding thioesterases with varying substrate preferences have been identified from plant seeds. These genes or functional subsequences thereof can be used to engineer organisms to produce fatty acids having a chain-length distribution (fatty acid profile) that is altered from the wild type organism. Specifically, recombinant cells express one or more of the exogenous FatB genes. The fatty acids can be further converted to triglycerides, fatty aldehydes, fatty alcohols and other oleochemicals either synthetically or biosynthetically. In specific embodiments, triglycerides are produced by a host cell expressing the novel FatB gene. A triglyceride-containing natural oil can be recovered from the host cell. The natural oil can be refined, degummed, bleached and/or deodorized. The oil, in its natural or processed form, can be used for foods, chemicals, fuels, cosmetics, plastics, and other uses.
The genes can be used in a variety of genetic constructs including plasmids or other vectors for expression or recombination in a host cell. The genes can be codon optimized for expression in a target host cell. The proteins produced by the genes can be used in vivo or in purified form.
The gene sequences disclosed can also be used to prepare antisense, or inhibitory RNA (e.g., RNAi or hairpin RNA) to inhibit complementary genes in a plant or other organism.
FatB genes found to be useful in producing desired fatty acid profiles in a cell are summarized below in Table 1. Nucleic acids or proteins having the sequence of SEQ ID NOS: 1-78 can be used to alter the fatty acid profile of a recombinant cell. Variant nucleic acids can also be used; e.g, variants having at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, or 78. Codon optimization of the genes for a variety of host organisms is contemplated, as is the use of gene fragments. Preferred codons for Prototheca strains and for Chlorella protothecoides are shown below in Tables 2 and 3, respectively. In some embodiments, the first and/or second most preferred Prototheca codons are employed for codon optimization.
In embodiments of the invention, there is protein or a nucleic acid encoding a protein having any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, or 77. In an embodiment, there is protein or a nucleic acid encoding a protein having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with any of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, or 77. In certain embodiments, the invention encompasses a fragment any of the above-described proteins or nucleic acids (including fragments of protein or nucleic acid variants), wherein the protein fragment has acyl-ACP thioesterase activity or the nucleic acid fragment encodes such a protein fragment. In other embodiments, the fragment includes a domain of an acyl-ACP thioesterase that mediates a particular function, e.g., a specificity-determining domain. Illustrative fragments can be produced by C-terminal and/or N-terminal truncations and include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length sequences disclosed herein.
The term “percent sequence identity,” in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties; Gap×drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap×drop-off 50; Expect: 10; Word Size: 3; Filter: on.
In certain embodiments, percent sequence identity for variants of the nucleic acids or proteins discussed above can be calculated by using the full-length nucleic acid sequence (e.g., one of SEQ ID NOS: 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47, 48, 50, 51, 53, 54, 56, 57, 59, 60, 62, 63, 65, 66, 68, 69, 71, 72, 74, 76, or 78) or full-length amino acid sequence (e.g., one of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 75, or 77) as the reference sequence and comparing the full-length test sequence to this reference sequence. In some embodiments relating to fragments, percent sequence identity for variants of nucleic acid or protein fragments can be calculated over the entire length of the fragment.
The nucleic acids can be in isolated form, or part of a vector or other construct, chromosome or host cell. It has been found that is many cases the full length gene (and protein) is not needed; for example, deletion of some or all of the N-terminal hydrophobic domain (typically an 18 amino acid domain starting with LPDW (SEQ ID NO: 115)) yields a still-functional gene. In addition, fusions of the specificity determining regions of the genes in Table 1 with catalytic domains of other acyl-ACP thioesterases can yield functional genes. Thus, in certain embodiments, the invention encompasses functional fragments (e.g., specificity determining regions) of the disclosed nucleic acid or amino acids fused to heterologous acyl-ACP thioesterase nucleic acid or amino acid sequences, respectively.







TABLE 1







FatB genes according to embodiments of the present invention
















Native CDS
Prototheca






nucloetide
moriformis




Sequence
Amino Acid
sequence (not
codon-




Variant (relative
Sequence of
codon-
optimized




to dominant
CDS (no
optimized, no
nucleotide




transcript
additional
additional
sequence


Species
Gene Name
idenitified)
tags)
cloning sites)
of CDS





Cinnamomum
CcFATB1b
M25L, M322R,
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID


camphora

ΔT367-D368


NO: 3


Cinnamomum
CcFATB4
“wild-type”
SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID


camphora




NO: 6


Cinnamomum
CcFATB3
“wild-type”
SEQ ID NO: 7
SEQ ID NO: 8
SEQ ID


camphora




NO: 9


Cuphea
ChsFATB1
“wild-type”
SEQ ID NO:
SEQ ID NO: 11
SEQ ID


hyssopifolia


10

NO: 12


Cuphea
ChsFATB2
“wild-type”
SEQ ID NO:
SEQ ID NO: 14
SEQ ID


hyssopifolia


13

NO: 15


Cuphea
ChsFATB2b
+a.a.248-259
SEQ ID NO:
SEQ ID NO: 17
SEQ ID


hyssopifolia


16

NO: 18


Cuphea
ChsFATB3
“wild-type”
SEQ ID NO:
SEQ ID NO: 20
SEQ ID


hyssopifolia


19

NO: 21


Cuphea
ChsFATB3b
V204I, C239F,
SEQ ID NO:
SEQ ID NO: 23
SEQ ID


hyssopifolia

E243D, M251V
22

NO: 24


Cuphea
CuPSR23FATB3
“wild-type”
SEQ ID NO:
SEQ ID NO: 26
SEQ ID


PSR23


25

NO: 27


Cuphea
CwFATB3
“wild-type”
SEQ ID NO:
SEQ ID NO: 29
SEQ ID


wrightii


28

NO: 30


Cuphea
CwFATB4a
“wild-type”
SEQ ID NO:
SEQ ID NO: 32
SEQ ID


wrightii


31

NO: 33


Cuphea
CwFATB4b
“wild-type”
SEQ ID NO:
SEQ ID NO: 35
SEQ ID


wrightii


34

NO: 36


Cuphea
CwFATB5
“wild-type”
SEQ ID NO:
SEQ ID NO: 38
SEQ ID


wrightii


37

NO: 39


Cuphea
ChtFATB1a
“wild-type”
SEQ ID NO:
SEQ ID NO: 41
SEQ ID


heterophylla


40

NO: 42


Cuphea
ChtFATB1b
P16S, T20P, G94S,
SEQ ID NO:
SEQ ID NO: 44
SEQ ID


heterophylla

G105W, S293F,
43

NO: 45




L305F


Cuphea
ChtFATB2b
“wild-type”
SEQ ID NO:
SEQ ID NO: 47
SEQ ID


heterophylla


46

NO: 48


Cuphea
ChtFATB2a
S17P, P21S, T28N,
SEQ IDO NO:
SEQ ID NO: 50
SEQ ID


heterophylla

L30P, S33L,
49

NO: 51




G76D, S78P,




G137W


Cuphea
ChtFATB2c
G76D, S78P
SEQ ID NO:
SEQ ID NO: 53
SEQ ID


heterophylla


52

NO: 54


Cuphea
ChtFATB2d
S21P, T28N,
SEQ ID NO:
SEQ ID NO: 56
SEQ ID


heterophylla

L30P, S33L,
55

NO: 57




G76D, R97L,




H124L, W127L,




I132S, K258N,




C303R, E309G,




K334T, T386A


Cuphea
ChtFATB2e
G76D, R97L,
SEQ ID NO:
SEQ ID NO: 59
SEQ ID


heterophylla

H124L, I132S,
58

NO: 60




G152S, H165L,




T211N, K258N,




C303R, E309G,




K334T, T386A


Cuphea
ChtFATB2f
R97L, H124L,
SEQ ID NO:
SEQ ID NO: 62
SEQ ID


heterophylla

I132S, G152S,
61

NO: 63




H165L, T211N


Cuphea
ChtFATB2g
A6T, A16V, S17P,
SEQ ID NO:
SEQ ID NO: 65
SEQ ID


heterophylla

G76D, R97L,
64

NO: 66




H124L, I132S,




S143I, G152S,




A157T, H165L,




T211N, G414A


Cuphea
ChtFATB3a
“wild-type”
SEQ ID NO:
SEQ ID NO: 68
SEQ ID


heterophylla


67

NO: 69


Cuphea
ChtFATB3b
C67G, H72Q,
SEQ ID NO:
SEQ ID NO: 71
SEQ ID


heterophylla

L128F, N179I
70

NO: 72


Cuphea
CvisFATB1
published
SEQ ID NO:
N/A
SEQ ID


viscosissima


73

NO: 74


Cuphea
CvisFATB2
published
SEQ ID NO:
N/A
SEQ ID


viscosissima


75

NO: 76


Cuphea
CvisFATB3
published
SEQ ID NO:
N/A
SEQ ID


viscosissima


77

NO: 78
















TABLE 2





Preferred codon usage in Prototheca strains




















Ala
GCG
345 (0.36)
Asn
AAT
  8 (0.04)



GCA
 66 (0.07)

AAC
201 (0.96)



GCT
101 (0.11)
Pro
CCG
161 (0.29)



GCC
442 (0.46)

CCA
 49 (0.09)


Cys
TGT
 12 (0.10)

CCT
 71 (0.13)



TGC
105 (0.90)

CCC
267 (0.49)


Asp
GAT
 43 (0.12)
Gln
CAG
226 (0.82)



GAC
316 (0.88)

CAA
 48 (0.18)


Glu
GAG
377 (0.96)
Arg
AGG
 33 (0.06)



GAA
 14 (0.04)

AGA
 14 (0.02)


Phe
TTT
 89 (0.29)

CGG
102 (0.18)



TTC
216 (0.71)

CGA
 49 (0.08)


Gly
GGG
 92 (0.12)

CGT
 51 (0.09)



GGA
 56 (0.07)

CGC
331 (0.57)



GGT
 76 (0.10)
Ser
AGT
 16 (0.03)



GGC
559 (0.71)

AGC
123 (0.22)


His
CAT
 42 (0.21)

TCG
152 (0.28)



CAC
154 (0.79)

TCA
 31 (0.06)


Ile
ATA
  4 (0.01)

TCT
 55 (0.10)



ATT
 30 (0.08)

TCC
173 (0.31)



ATC
338 (0.91)
Thr
ACG
184 (0.38)


Lys
AAG
284 (0.98)

ACA
 24 (0.05)



AAA
  7 (0.02)

ACT
 21 (0.05)


Leu
TTG
 26 (0.04)

ACC
249 (0.52)



TTA
  3 (0.00)
Val
GTG
308 (0.50)



CTG
447 (0.61)

GTA
  9 (0.01)



CTA
 20 (0.03)

GTT
 35 (0.06)



CTT
 45 (0.06)

GTC
262 (0.43)



CTC
190 (0.26)
Trp
TGG
107 (1.00)


Met
ATG
191 (1.00)
Tyr
TAT
 10 (0.05)






TAC
180 (0.95)





Stop
TGA/TAG/TAA
















TABLE 3





Preferred codon usage in Chlorella protothecoides




















TTC (Phe)
TAC (Tyr)
TGC (Cys)
TGA (Stop)


 



TGG (Trp)
CCC (Pro)
CAC (His)
CGC (Arg)


 



CTG (Leu)
CAG (Gln)
ATC (Ile)
ACC (Thr)


 



GAC (Asp)
TCC (Ser)
ATG (Met)
AAG (Lys)


 



GCC (Ala)
AAC (Asn)
GGC (Gly)
GTG (Val)


 



GAG (Glu)












Host Cells

The host cell can be a single cell or part of a multicellular organism such as a plant. Methods for expressing Fatb genes in a plant are given in U.S. Pat. Nos. 5,850,022; 5,723,761; 5,639,790; 5,807,893; 5,455,167; 5,654,495; 5,512,482; 5,298,421; 5,667,997; and 5,344,771; 5,304,481, or can be accomplished using other techniques generally known in plant biotechnology. Engineering of oleaginous microbes including Chlorophyta is disclosed in WO2010/063032, WO2011,150411, and WO2012/106560 and in the examples below.
Examples of oleaginous host cells include plant cells and microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae. Specific examples of microalgal cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae are provided in Published PCT Patent Applications WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/150411, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cell weight, ±5%. Optionally, the oils produced can be low in DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose). In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylose metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, “GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE”, published Nov. 15, 2012.
Oils and Related Products
The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature natural oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.
The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. WO2008/151149, WO2010/06032, WO2011/150410, and WO2011/1504 disclose heterotrophic cultivation and oil isolation techniques. For example, oil may be obtained by cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. After recovery of the oil, a valuable residual biomass remains. Uses for the residual biomass include the production of paper, plastics, absorbents, adsorbents, as animal feed, for human nutrition, or for fertilizer.
Where a fatty acid profile of a triglyceride (also referred to as a “triacylglyceride” or “TAG”) cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell.
The stable carbon isotope value δ13C is an expression of the ratio of 13C/12C relative to a standard (e.g. PDB, carbonite of fossil skeleton of Belemnite americana from Peedee formation of South Carolina). The stable carbon isotope value δ13C (0/00) of the oils can be related to the δ13C value of the feedstock used. In some embodiments, the oils are derived from oleaginous organisms heterotrophically grown on sugar derived from a C4 plant such as corn or sugarcane. In some embodiments the δ13C (0/00) of the oil is from −10 to −17 0/00 or from −13 to −16 0/00.
The oils produced according to the above methods in some cases are made using a microalgal host cell. As described above, the microalga can be, without limitation, fall in the classification of Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol, when detected by GC-MS. However, it is believed that all sterols produced by Chlorella have C24β stereochemistry. Thus, it is believed that the molecules detected as campesterol, stigmasterol, and β-sitosterol, are actually 22,23-dihydrobrassicasterol, proferasterol and clionasterol, respectively. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24β stereochemistry and the absence of C24α stereochemistry in the sterols present. For example, the oils produced may contain 22,23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in β-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of Δ7-poriferasterol.
In embodiments of the present invention, oleaginous cells expressing one or more of the genes of Table 1 can produce an oil with at least 20% of C8, C10, C12, C14 or C16 fatty acids. In a specific embodiment, the level of myristate (C14:0) in the oil is greater than 30%.
Thus, in embodiments of the invention, there is a process for producing an oil, triglyceride, fatty acid, or derivative of any of these, comprising transforming a cell with any of the nucleic acids discussed herein. In another embodiment, the transformed cell is cultivated to produce an oil and, optionally, the oil is extracted. Oil extracted in this way can be used to produce food, oleochemicals or other products.
The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc). The oils, triglycerides, fatty acids from the oils may be subjected to C—H activation, hydroamino methylation, methoxy-carbonation, ozonolysis, enzymatic transformations, epoxidation, methylation, dimerization, thiolation, metathesis, hydro-alkylation, lactonization, or other chemical processes.
After extracting the oil, a residual biomass may be left, which may have use as a fuel, as an animal feed, or as an ingredient in paper, plastic, or other product. For example, residual biomass from heterotrophic algae can be used in such products.
The described embodiments of the invention are intended to be merely exemplary and numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention. For example, the various triglyceride oils can be tailored in for a mixture of midchain and long chain fatty acids in order to adjust parameters such as polarity, solvency, and foam-height of the oils or chemicals made from the oils.
EXAMPLE 1
Sequences of novel plant acyl-ACP thioesterases involved in seed-specific midchain (C8-C16) fatty acid biosynthesis in higher plants were isolated. Seed-specific lipid production genes were isolated through direct interrogation of RNA pools accumulating in oilseeds. Based on phylogenetic analysis, novel enzymes can be classified as members of FatB family of acyl-ACP thioesterases.
Seeds of oleaginous plants were obtained from local grocery stores or requested through USDA ARS National Plant Germplasm System (NPGS) from North Central Regional Plant Introduction Station (NCRIS) or USDA ARS North Central Soil Conservation Research Laboratory (Morris, Mich.). Dry seeds were homogenized in liquid nitrogen to powder, resuspended in cold extraction buffer containing 6-8M Urea and 3M LiCl and left on ice for a few hours to overnight at 4° C. The seed homogenate was passed through NucleoSpin Filters (Macherey-Nagel) by centrifugation at 20,000 g for 20 minutes in the refrigerated microcentrifuge (4° C.). The resulting RNA pellets were resuspended in the buffer containing 20 mM Tris HCl, pH7.5, 0.5% SDS, 100 mM NaCl, 25 mM EDTA, 2% PVPP) and RNA was subsequently extracted once with Phenol-Chloroform-Isoamyl Alcohol (25:24:1, v/v) and once with chloroform. RNA was finally precipitated with isopropyl alcohol (0.7 Vol.) in the presence of 150 mM of Na Acetate, pH5.2, washed with 80% ethanol by centrifugation, and dried. RNA samples were treated with Turbo DNAse (Lifetech) and purified further using RNeasy kits (Qiagen) following manufacturers' protocols. The resulting purified RNA samples were converted to pair-end cDNA libraries and subjected to next-generation sequencing (2×100 bp) using Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using Trinity or Oases packages. Putative thioesterase-containg cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases. These in silico identified putative thioesterase cDNAs have been further verified by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting full-length thioesterase cDNAs. The resulting amplified products were cloned and sequenced de novo to confirm authenticity of identified thioesterase genes.
To interrogate evolutionary and functional relationship between novel acyl-ACP thioesterases and the members of two existing thioesterase classes (FatA and FatB), we performed a phylogenetic analysis using published full-length (Mayer and Shanklin, 2007) and truncated (THYME database) amino acid thioesterase sequences. Novel proteins appear to group with known acyl-ACP FatB thioesterases involved in biosynthesis of C8-C16 fatty acids. Moreover, novel thioesterases appear to cluster into 3 predominant out-groups suggesting distinct functional similarity and evolutionary relatedness among members of each cluster.
The amino acid sequences of the FatB genes follow are shown in Table 4.







TABLE 4





Amino acid sequences of FatB genes















CuPSR23 FATB3 (SEQ ID NO: 25):


MVVAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNAGFQVKANASAH


 


PKANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLD


 


RKSKRPDMLVDSVGLKCIVRDGLVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCK


 


SLGLLNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASD


 


WLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLH


 


KFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRR


 


ECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTST


 


AKTSNGNSVS


 


CuPSR23 FATB3b (SEQ ID NO: 79):


MVVAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNAGFQVKANASAH


 


PKANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAITTVFVAAEKQWTMLD


 


RKSKRPDMLVDSVGLKSIVRDGLVSRQSFLIRSYEIGADRTASIETLMNHLQETSINHCKS


 


LGLLNDGFGRTPGMCKNDLIWVLTKMQIMVNRYPTWGDTVEINTWFSQSGKIGMASD


 


WLISDCNTGEILIRATSVWAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLH


 


KFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRR


 


ECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNAGTNGAISTST


 


AKTSNGNSAS


 


CwFATB3 (SEQ ID NO: 28):


MVVAAAASSAFFPVPAPRTTPKPGKFGNWPSSLSPPFKPKSNPNGRFQVKANVSP


 


HPKANGSAVSLKSGSLNTLEDPPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLD


 


RKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCK


 


SVGLLNDGFGRTSEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGRD


 


WLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSICKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRR


 


ECGRESVVESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNRAIST


 


CwFATB3a (SEQ ID NO: 28):


MVVAAAASSAFFPVPAPRTTPKPGKFGNWPSSLSPPFKPKSNPNGRFQVKANVSP


 


HPKANGSAVSLKSGSLNTLEDPPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLD


 


RKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCK


 


SVGLLNDGFGRTSEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGRD


 


WLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSICKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRR


 


ECGRESVVESVTSMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNRAIST


 


CwFATB3b (SEQ ID NO: 80):


MVVAAAASSAFFPVPAPRTTPKPGKFGNWPSSLSPPFKPKSNPNGRFQVKANVSP


 


HPKANGSAVSLKSGSLNTLEDLPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQFTRLD


 


RKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCK


 


SVGLLNDGFGRTSEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGRD


 


WLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSICKGLTPGWNDLDVNQHVSNVKYIGWILEKFWRPRSYALSPLNIGGNVE


 


GKVW


 


CwFATB3c (SEQ ID NO: 81):


MVVAAAASSAFFPVPAPRTTPKPGKFGNWPSSLSPPFKPKSNPNGRFQVKANVSP


 


HPKANGSAVSLKSGSLNTLEDLPSSPPPRTFLNQLPDWSRLRTAITTVFVATEKQFTRLD


 


RKSKRPDMLVDWFGSETIVQDGLVFRERFSIRSYEIGADRTASIETLMNHLQDTSLNHCK


 


SVGLLNDGFGRTSEMCTRDLIWVLTKMQIVVNRYPTWGDTVEINSWFSQSGKIGMGRD


 


WLISDCNTGEILVRATSAWAMMNQKTRRFSKLPCEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSICKGLTPGWNDLDVNQHVSNVKYIGWILEKFWRPRSYALSPLNIGGNVE


 


GKVW


 


CwFATB4a (SEQ ID NO: 31):


MVATAASSAFFPVPSADTSSSRPGKLGSGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGGFKTQEDSPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSNDGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEYR


 


RECGRESVLESLTAVDPSAEGYASRFQHLLRLEDGGEIVKARTEWRPKNAGINGVVPSE


 


ESSPGDFF


 


CwFATB4a.1 (SEQ ID NO: 82):


MVATAASSAFFPVPSADTSSSRPGKLGSGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGGFKTQEDSPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSNDGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEYR


 


RECGRESVLESLTAVDPSAEGYASRFQHLLRLEDGGEIVKARTEWRPKNAGINWVVPSE


 


ESSPGDFF


 


CwFATB4a.2 (SEQ ID NO: 83):


MVATAASSAFFPVPSADTSSSRPGKLGNGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGSFKTQEDAPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSNDGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEYR


 


RECGRESVLESLTAVDPSAEGYASRFQHLLRLEDGGEIVKARTEWRPKNAGINGVVPSE


 


ESSPGDFF


 


CwFATB4a.3 (SEQ ID NO: 84):


MVATAASSAFFPVPSADTSSSRPGKLGSGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGGFKTQEDSPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSNDGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEYR


 


RECGRESVLESLTAVDPSAEGYVSRFQHLLRLEDGGEIVKARTEWRPKNAGINGVVPSE


 


ESSPGDFF


 


CwFATB4b (SEQ ID NO: 34):


MVATAASSAFFPVPSADTSSSRPGKLGNGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGSFKTQEDAPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSSDGFGRTPAMSKRDLIWVVAKMQVMVNRYPAWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPAEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSGEGDGSKFQHLLRLEDGGEIVKARTEWRPKNAGINGVVPS


 


EESSPGGDFF


 


CwFATB4b.1 (SEQ ID NO: 85):


MVATAASSAFFPVPSADTSSSRPGKLGSGPSSLSPLKPKSIPNGGLQVKANASAPP


 


KINGSSVGLKSGSFKTQEDAPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGSIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKI


 


AGLSSDGFGRTPAMSKRDLIWVVAKMQVMVNRYPAWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRNEIEPHFVDSAPVVEDDDRK


 


LPKLDENTADSIRKGLTPRWNDLDVNQHVNNVKYIGWILESTPAEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSGEGDGSKFQHLLRLEDGGEIVKARTEWRPKNAGINGVVPS


 


EESSPGGDFF


 


CwFATB5 (SEQ ID NO: 37):


MVAAAASSAFFSVPTPGTPPKPGKFGNWPSSLSVPFKPDNGGFHVKANASAHPK


 


ANGSAVNLKSGSLETPPRSFINQLPDLSVLLSKITTVFGAAEKQWKRPGMLVEPFGVDRI


 


FQDGVFFRQSFSIRSYEIGVDRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRD


 


LIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCRTGEILIRATSVWA


 


MMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDQKLQKLDVKTGDSIRDGLTPRWND


 


LDVNQHVNNVKYIGWILKSVPIEVFETQELCGVTLEYRRECGRDSVLESVTAMDPAKEG


 


DRCVYQHLLRLEDGADITIGRTEWRPKNAGANGAMSSGKTSNGNCLIEGRGWQPFRVV


 


RLIF


 


CwFATB5a (SEQ ID NO: 86):


MVAAAASSAFFSVPTPGTPPKPGKFGNWPSSLSVPFKPDNGGFHVKANASAHPK


 


ANGSAVNLKSGSLETPPRSFINQLPDLSVLLSKITTVFGAAEKQWKRPGMLVEPFGVDRI


 


FQDGFFFRQSFSIRSYEIGVDRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRD


 


LIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCRTGEILIRATSVWA


 


MMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDQKLQKLDVKTGDSIRDGLTPRWND


 


LDVNQHVNNVKYIGWILKSVPIEVFETQELCGVTLEYRRECGRDSVLESVTAMDPAKEG


 


DRCVYQHLLRLEDGADITIGRTEWRPKNAGANGAMSSGKTSNGNCLIEGRGWQPFRVV


 


RLIF


 


CwFATB5b (SEQ ID NO: 87):


MVAAAASSAFFSVPTPGTPPKPGKFGNWPSSLSVPFKPDNGGFHVKANASAHPK


 


ANGSAVNLKSGSLETPPRSFINQLPDLSVLLSKITTVFGAAEKQWKRPGMLVEPFGVDRI


 


FQDGVFFRQSFSIRSYEIGVDRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRD


 


LIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCRTGEILIRATSVWA


 


MMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDQKLQKLDVKTGDSIRDGLTPRWND


 


LDVNQHVNNVKYIGWILKSVPIEVFETQELCGVTLEYRRECGRDSVLESVTAMDPAKEG


 


DRCVYQHLLWLEDGADITIGRTEWRPKNAGANGAMSSGKTSNGNCLIEGRGWQPFRV


 


VRLIF


 


CwFATB5c (SEQ ID NO: 88):


MVAAAASSAFFSVPTPGTPPKPGKFGNWPSSLSVPFKPDNGGFHVKANASAHPK


 


ANGSAVNLKSGSLETPPRSFINQLPDLSVLLSKITTVFGAAEKQWKRPGMLVEPFGVDRI


 


FQDGVFFRQSFSIRSYEIGVDRTASIETLMNIFQETSLNHCKSIGLLNDGFGRTPEMCKRD


 


LIWVVTKIQVEVNRYPIWGDTIEVNTWVSESGKNGMGRDWLISDCRTGEILIRATSVWA


 


MMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDQKLQKLDVKTGDSIRDGLTPRWND


 


LDVNQHVNNVKYIGWILKSVPIEVFETQELCGVTLEYRRECGRDSVLESVTAMDPAKEG


 


DRCVYQHLLRLEDGADITIGRTEWRPKNAGANGAMSSGKTSNGNCLIEGMGWQPFRVV


 


RLIF


 


CwFATB5.1 (SEQ ID NO: 89):


MVAAAASSAFFSVPTPGTSPKPGKFRNWPSSLSVPFKPETNHNGGFHIKANASAH


 


PKANGSALNLKSGSLETQEDTSLSSPPRTFIKQLPDWSMLLSKITTVFGAAEKQLKRPGM


 


LVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLNDGFG


 


RTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCRTGE


 


ILIRATSVWAMMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDRKLYKLNVKTGDSIR


 


DGLTPRWNDLDVNQHVNNVKFIGWILKSVPTKVFETQELCGVTLEYRRECGKDSVLES


 


VTAMDPAKEGDRSVYQHLLRLEDGADITIGRTEWRPKNAGANEAISSGKTSNGNSAS


 


CwFATB5.1a (SEQ ID NO: 90):


MVAAAASSAFFSVPTPGTSPKPGKFRNWPLSLSVPFKPETNHNGGFHIKANASAH


 


PKANGSALNLKSGSLETQEDTSLSSPPRTFIKQLPDWSMLLSKITTVFGAAEKQLKRPGM


 


LVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLNDGFG


 


RTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCRTGE


 


ILIRATSVWAMMNQNTRRLSKFPYEVRQEIAPHFVDSAPVIEDDRKLYKLNVKTGDSIR


 


DGLTPRWNDLDVNQHVNNVKFIGWILKSVPTKVFETQELCGVTLEYRRECGKDSVLES


 


VTAMDPAKEGDRSVYQHLLRLEDGADITIGRTEWRPKNAGANEAISSGKTSNGNSAS


 


CcFATB2b (SEQ ID NO: 91):


MVTTSLASAYFSMKAVMLAPDGRGIKPRSSGLQVRAGNERNSCKVINGTKVKD


 


TEGLKGCSTLQGQSMLDDHFGLHGLVFRRTFAIRCYEVGPDRSTSIMAVMNHLQEAAR


 


NHAESLGLLGDGFGETLEMSKRDLIWVVRRTHVAVERYPAWGDTVEVEAWVGASGNT


 


GMRRDFLVRDCKTGHILTRCTSVSVMMNMRTRRLSKIPQEVRAEIDPLFIEKVAVKEGEI


 


KKLQKLNDSTADYIQGGWTPRWNDLDVNQHVNNIIYVGWIFKSVPDSISENHHLSSITLE


 


YRRECIRGNKLQSLTTVCGGSSEAGIICEHLLQLEDGSEVLRARTEWRPKHTDSFQGISER


 


FPQQEPHK


 


CcFATB3 (SEQ ID NO: 7):


MVATAAASAFFPVGAPATSSATSAKASMMPDNLDARGIKPKPASSSGLQVKAN


 


AHASPKINGSKVSTDTLKGEDTLTSSPAPRTFINQLPDWSMFLAAITTIFLAAEKQWTNL


 


DWKPRRPDMLADPFGIGRFMQDGLIFRQHFAIRSYEIGADRTASIETLMNHLQETALNH


 


VRSAGLLGDGFGATPEMSRRDLIWVVTRMQVLVDRYPAWGDIVEVETWVGASGKNG


 


MRRDWLVRDSQTGEILTRATSVWVMMNKRTRRLSKLPEEVRGEIGPYFIEDVAIIEEDN


 


RKLQKLNENTADNVRRGLTPRWSDLDVNQHVNNVKYIGWILESAPGSILESHELSCMTL


 


EYRRECGKDSVLQSMTAVSGGGSAAGGSPESSVECDHLLQLESGPEVVRGRTEWRPKS


 


ANNSRSILEMPAESL


 


CcFATB3b (SEQ ID NO: 92):


MVATAAASAFFPVGAPATSSATSAKASMMPDNLDARGIKPKLASSSGLQVKAN


 


AHASPKINGSKVSTDTLKGEDTLTSSPAPRTFINQLPDWSMFLAAITTIFLAAEKQWTNL


 


DWKPRRPDMLADPFGIGRFMQDGLIFRQHFAIRSYEIGADRTASIETLMNHLQETALNH


 


VRSAGLLGDGFGATPEMSRRDLIWVVTRMQVLVDRYPAWGDIVEVETWVGASGKNG


 


MRRDWLVRDSQTGEILTRATSVWVMMNKRTRRLSKLPEEVRGEIGPYFIEDVAIIEEDN


 


RKLQKLNENTADNVRRGLTPRWSDLDVNQHVNNVKYIGWILESAPGSILESHELSCMTL


 


EYRRECGKDSVLQSMTAVSGGGSAAGGSPESSVECDHLLQLESGPEVVRGRTEWRPKS


 


ANNSRSILEMPAESL


 


CcFATB3c (SEQ ID NO: 93):


MVATAAASAFFPVGAPATSSATSAKASMMPDNLDARGIKPKPASSSGLQVKAN


 


AHASPKINGSKVSTDTLKGEDTLTSSPAPRTFINQLPDWSMFLAAITTIFLAAEKQWTNL


 


DWKPRRPDMLADPFGIGRFMQDGLIFRQHFAIRSYEIGADRTASIETLMNHLQETALNH


 


VRSAGLLGDGFGATPEMSRRDLIWVVTRMQVLVDRYPAWGDIVEVETWVGASGKNG


 


MRRDWLVRDSQTGEILTRATSVWVMMNKRTRRLSKLPEEVRGEIGPYFIEDVAIIEEDN


 


RKLQKLNENTADNVRRGLTPRWSDLDVNQHVNNAKYIGWILESAPGSILESHELSCMTL


 


EYRRECGKDSVLQSMTAVSGGGSAAGGSPESSVECDHLLQLESGPEVVRGRTEWRPKS


 


ANNSRSILEMPAESL


 


ChtFATB1a (SEQ ID NO: 40):


MVAAAASSAFFSVPTPGTSTKPGNFGNWPSSLSVPFKPESNHNGGFRVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWGMLLSKITTVFGAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCR


 


TGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTGD


 


SIRKGLTPRWNDLDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDSVL


 


ESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGANGAISTGKTSNENSVS


 


ChtFATB1a.1 (SEQ ID NO: 94):


MVAAAASSAFFSVPTPGTSPKPGNFGNWPSSLSVPFKPESNHNGGFRVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWGMLLSKITTVFGAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRHSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLIGDC


 


RTGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTG


 


DSIRKGLTPRWNDLDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDS


 


VLESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGANGALSTGKTSNGN


 


SVS


 


ChtFATB1a.2 (SEQ ID NO: 95):


MVAAAASSAFFSVPTPGTSPKPGNFGNWPSNLSVPFKPESNHNGGFRVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWGMLLSKITTVFGAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCR


 


TGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTGD


 


SIRKGLTPRWNDFDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDSVL


 


ESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGANGAISTGKTSNENSVS


 


ChtFATB1a.3 (SEQ ID NO: 96):


MVAAAASSAFFSVPTPGTSPKPGNFGNWPSSLSVPFKPESNHNGGFRVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWGMLLSKITTVFGAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCR


 


TGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTGD


 


SIRKGLTPRWNDFDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDSVL


 


ESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGVNGAISTGKTSNENSVS


 


ChtFATB1a.4 (SEQ ID NO: 97):


MVAAAASSAFFSVPTPGTSPKPGNFGNWPSSLSVPFKPESNHNGGFRVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWSMLLSKITTVFGAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCR


 


TGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTGD


 


SIRKGLTPRWNDFDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDSVL


 


ESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGANGAISTGKTSNENSVS


 


ChtFATB1b (SEQ ID NO: 43):


MVAAAASSAFFSVPTSGTSPKPGNFGNWPSSLSVPFKPESSHNGGFQVKANASA


 


HPKANGSAVNLKSGSLETQEDTSSSSPPPRTFIKQLPDWSMLLSKITTVFWAAERQWKRP


 


GMLVEPFGVDRIFQDGVFFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHCKSIGLLND


 


GFGRTPEMCKRDLIWVVTKIQVEVNRYPTWGDTIEVNTWVSESGKNGMGRDWLISDCR


 


TGEILIRATSVWAMMNRKTRRLSKFPYEVRQEIAPHFVDSAPVIEDDKKLHKLDVKTGD


 


FIRKGLTPRWNDFDVNQHVNNVKYIGWILKSVPAEVFETQELCGVTLEYRRECGRDSVL


 


ESVTAMDTAKEGDRSLYQHLLRLEDGADITIGRTEWRPKNAGANGAISTGKTSNENSVS


 


ChtFATB2b (SEQ ID NO: 46):


MVVAAAASSAFFPVPASGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTQEGTSSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQLTMLD


 


RKSKKPDMHVDWFGLEIIVQDGLVFRESFSIRSYEIGADRTASIETLMNHLQDTSLNHCK


 


SVGLLNDGFGRTPEMCKRDLIWVLTKMQIMVNRYPTWGDTVEINSWFSQSGKIGMGRN


 


WLISDCNTGEILIRATSIWAMMNQKTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLHK


 


FDVKTGDSICKGLTPEWNDLDVNQHVSNVKYIGWILESMPKEVLDTQELCSLTLEYRRE


 


CGRDSVLESVTAMDPSKVGDRSQYQHLLRLEDGTDIMKGRTEWRPKNAGTNGAISTGK


 


TSNGNSVS


 


ChtFATB2a (SEQ ID NO: 49):


MVVAAAASSAFFPVPAPGTTSKPGKFGNWPSSLSPSFKPKSNPNGGFQVKANAS


 


AHPKANGSAVSLKSGSLNTKEDTPSSPPPRTFLNQLPDWSRLRTAITTVFVAAEKQLTML


 


DRKSKKPDMHVDWFGLEIIVQDWLVFRESFSIRSYEIGADRTASIETLMNHLQDTSLNHC


 


KSVGLLNDGFGRTPEMCKRDLIWVLTKMQIMVNRYPTWGDTVEINSWFSQSGKIGMGR


 


NWLISDCNTGEILIRATSIWAMMNQKTRRFSKLPNEVRQEIAPHFVDAPPLIEDNDRKLH


 


KFDVKTGDSICKGLTPEWNDLDVNQHVSNVKYIGWILESMPKEVLDTQELCSLTLEYRR


 


ECGRDSVLESVTAMDPSKVGDRSQYQHLLRLEDGTDIMKGRTEWRPKNAGTNGAISTG


 


KTSNGNSVS


 


ChtFATB2c (SEQ ID NO: 52):


MVVAAAASSAFFPVPASGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTKEDTPSSPPPRTFLNQLPDWNRLRTAITTVFVAAEKQLTML


 


DRKSKKPDMHVDWFGLEIIVQDGLVFRESFSIRSYEIGADRTASIETLMNHLQDTSLNHC


 


KSVGLLNDGFGRTPEMCKRDLIWVLTKMQIMVNRYPTWGDTVEINSWFSQSGKIGMGR


 


NWLISDCNTGEILIRATSIWAMMNQKTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSICKGLTPEWNDLDVNQHVSNVKYIGWILESMPKEVLDTQELCSLTLEYRR


 


ECGRDSVLESVTAMDPSKVGDRSQYQHLLRLEDGTDIMKGRTEWRPKNAGTNGAISTG


 


KTSNGNSVS


 


ChtFATB2d (SEQ ID NO: 55):


MVVAAAASSAFFPVPAPGTTSKPGKFGNWPSSLSPSFKPKSNPNGGFQVKANAS


 


AHPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLNQLPDWSRLLTAISTVFVAAEKQLTML


 


DRKSKRPDMLVDLFGLESIVQDGLVFRESYSIRSYEIGADRTASIETLMNHLQDTSLNHC


 


KSVGLLNDGFGRTPEMCKRDLIWVLTKMQIMVNRYPTWGDTVEINSWFSQSGKIGMGR


 


NWLISDCNTGEILIRATSIWAMMNQNTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLH


 


KFDVKTGDSIRKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRR


 


ECGRESVLESVTAMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNGAISTG


 


KTSNGNSVS


 


ChtFATB2e (SEQ ID NO: 58):


MVVAAAASSAFFPVPASGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTQEDTSSSPPPQTFLNQLPDWSRLLTAISTVFVAAEKQLTMLD


 


RKSKRPDMLVDWFGLESIVQDGLVFRESYSIRSYEISADRTASIETVMNLLQETSLNHCK


 


SMGILNDGFGRTPEMCKRDLIWVLTKMQILVNRYPNWGDTVEINSWFSQSGKIGMGRN


 


WLISDCNTGEILIRATSIWAMMNQNTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLHK


 


FDVKTGDSIRKGLTPGWNDLDVNQHVSNVKYIGWILESMPTEVLETQELCSLTLEYRRE


 


CGRDSVLESVTAMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGK


 


TSNGNSVS


 


ChtFATB2f (SEQ ID NO: 61):


MVVAAAASSAFFPVPASGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTQEGTSSSPPPRTFLNQLPDWSRLLTAISTVFVAAEKQLTMLD


 


RKSKRPDMLVDWFGLESIVQDGLVFRESYSIRSYEISADRTASIETVMNLLQETSLNHCK


 


SMGILNDGFGRTPEMCKRDLIWVLTKMQILVNRYPNWGDTVEINSWFSQSGKIGMGRN


 


WLISDCNTGEILIRATSIWAMMNQKTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLHK


 


FDVKTGDSICKGLTPEWNDLDVNQHVSNVKYIGWILESMPKEVLDTQELCSLTLEYRRE


 


CGRDSVLESVTAMDPSKVGDRSQYQHLLRLEDGTDIMKGRTEWRPKNAGTNGAISTGK


 


TSNGNSVS


 


ChtFATB2g (SEQ ID NO: 64):


MVVAATASSAFFPVPVPGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTQEDTSSSPPPRTFLNQLPDWSRLLTAISTVFVAAEKQLTMLD


 


RKSKRPDMLVDWFGLESIVQDGLVFREIYSIRSYEISADRTTSIETVMNLLQETSLNHCKS


 


MGILNDGFGRTPEMCKRDLIWVLTKMQILVNRYPNWGDTVEINSWFSQSGKIGMGRN


 


WLISDCNTGEILIRATSIWAMMNQKTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLHK


 


FDVKTGDSICKGLTPEWNDLDVNQHVSNVKYIGWILESMPKEVLDTQELCSLTLEYRRE


 


CGRDSVLESVTAMDPSKVGDRSQYQHLLRLEDGTDIMKGRTEWRPKNAGTNGAISTGK


 


TSNANSVS


 


ChtFATB2h (SEQ ID NO: 98):


MVVAAAASSAFFPVPASGTSPKPGKFGTWLSSSSPSYKPKSNPSGGFQVKANASA


 


HPKANGSAVSLKSGSLNTQEGTSSSPPPRTFLNQLPDWSRLLTAISTVFVAAEKQLTMLD


 


RKSKRPDMLVDWFGLESIVQDGLVFRESYSIRSYEISADRTASIETVMNLLQETSLNHCK


 


SMGILNDGFGRTPEMCKRDLIWVLTKMQILVNRYPNWGDTVEINSWFSQSGKIGMGRN


 


WLISDCNTGEILIRATSIWAMMNQNTRRFSKLPNEVRQEIAPHFVDAPPVIEDNDRKLHK


 


FDVKTGDSIRKGLTPGWNDLDVNQHVSNVKYIGWILESIPTEVLETQELCSLTLEYRREC


 


GRESVLESVTAMNPSKVGDRSQYQHLLRLEDGADIMKGRTEWRPKNAGTNGAISTGKT


 


SNGNSVS


 


ChtFATB3a (SEQ ID NO: 67):


MVATAASSAFFPVPSPDTSSRPGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSCSLKTHEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDWK


 


LPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSGKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIASG


 


ETSPGNS


 


ChtFATB3b (SEQ ID NO: 70):


MVATAASSAFFPVPSPDTSSRPGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGFGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLIEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMRR


 


DWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDWKL


 


PKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEYR


 


RECGRESVLESLTAVDPSGKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIASGE


 


TSPGNS


 


ChtFATB3c (SEQ ID NO: 99):


MVATAASSAFFPVPSPDTSSRPGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSCSLKTHEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDRK


 


LPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSEKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGAIAFG


 


ETSPGDS


 


ChtFATB3d (SEQ ID NO: 100):


MVATAASSAFFPVPSPDTSSRPGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSCSLKTHEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIKTVMNHLQETALNHVK


 


SAGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGM


 


RRDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDW


 


KLPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLE


 


YRRECGRESVLESLTAVDPSGKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIAS


 


GETSPGNS


 


ChtFATB3e (SEQ ID NO: 101):


MVATAASSAFFPVPSPDTSSRPGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSGSLKTHEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDWK


 


LPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSGKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIASG


 


ETSPGNS


 


ChtFATB3f (SEQ ID NO: 102):


MVATAASSAFFPVPSPDTSSRLGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMPVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDWK


 


LPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSEKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIASG


 


ETSPGNS


 


ChtFATB3g (SEQ ID NO: 103):


MVATAASSAFFPVPSPDTSSRAGKLGNGSSSLRPLKPKFVANAGLQVKANASAPP


 


KINGSSVSLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLDW


 


KPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHVKS


 


AGLLNEGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNGMR


 


RDWLISDCNTGEILTRASSVWVMMNQKTRKLSKIPDEVRHEIEPHFVDSAPVIEDDDWK


 


LPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTLEY


 


RRECGRESVLESLTAVDPSGKGFGPQFQHLLRLEDGGEIVKGRTEWRPKTAGINGTIASG


 


ETSPGNS


 


ChsFATB1 (SEQ ID NO: 10):


MVATNAAAFSAYTFFLTSPTHGYSSKRLADTQNGYPGTSLKSKSTPPPAAAAAR


 


NGALPLLASICKCPKKADGSMQLDSSLVFGFQFYIRSYEVGADQTVSIQTVLNYLQEAAI


 


NHVQSAGYFGDSFGATPEMTKRNLIWVITKMQVLVDRYPAWGDVVQVDTWTCSSGKN


 


SMQRDWFVRDLKTGDIITRASSVWVLMNRLTRKLSKIPEAVLEEAKLFVMNTAPTVDD


 


NRKLPKLDGSSADYVLSGLTPRWSDLDMNQHVNNVKYIAWILESVPQSIPETHKLSAIT


 


VEYRRECGKNSVLQSLTNVSGDGITCGNSIIECHHLLQLETGPEILLARTEWISKEPGFRG


 


APIQAEKVYNNK


 


ChsFATB2 (SEQ ID NO: 13):


MVATAASSAFFPVPSPDASSRPGKLGNGSSSLSPLKPKLMANGGLQVKANASAP


 


PKINGSSVGLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLD


 


WKPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHV


 


KSAGLLNDGFGRTLEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNG


 


MRRDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRHEIEPHFVDSAPVIEDDD


 


RKLPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTL


 


EYRRECGRESVLESLTAVDPSGKGSGSQFQHLLRLEDGGEIVKGRTEWRPKTAGINGPIA


 


SGETSPGDSS


 


ChsFatB2b (SEQ ID NO: 16):


MVATAASSAFFPVPSPDASSRPGKLGNGSSSLSPLKPKLMANGGLQVKANASAP


 


PKINGSSVGLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLD


 


WKPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHV


 


KSAGLLNDGFGRTLEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNG


 


MRRDWLISDCNTGEILTRASSKSQIMLPLHYCSVWVMMNQKTRRLSKIPDEVRHEIEPH


 


FVDSAPVIEDDDRKLPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPE


 


VLETQELCSLTLEYRRECGRESVLESLTAVDPSGKGSGSQFQHLLRLEDGGEIVKGRTEW


 


RPKTAGINGPIASGETSPGDSS


 


ChsFatB2c (SEQ ID NO: 104):


MVATAASSAFFPVPSPDASSRPGKLGNGSSSLSPLKPKLMANGGLQVKANASAP


 


PKINGSSVGLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLD


 


WKPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHV


 


KSAGLLNDGFGRTLEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNG


 


MRRDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRHEIEPHFVDSAPVIEDDD


 


RKLPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTL


 


EYRRECGRESVLESLTAVDPSGKGSGSQFQHLMRLEDGGEIVKGRTEWRPKTAGINGPI


 


ASGETSPGDSS


 


ChsFatB2d (SEQ ID NO: 105):


MVATAASSAFFPVPSPDASSRPGKLGNGSSSLSPLKPKLMANGGLQVKANASAP


 


PKINGSSVGLKSGSLKTQEDTPSAPPPRTFINQLPDWSMLLAAITTVFLAAEKQWMMLD


 


WKPKRPDMLVDPFGLGRIVQDGLVFRQNFSIRSYEIGADRTASIETVMNHLQETALNHV


 


KSAGLLNDGFGRTPEMYKRDLIWVVAKMQVMVNRYPTWGDTVEVNTWVAKSGKNG


 


MRRDWLISDCNTGEILTRASSVWVMMNQKTRRLSKIPDEVRHEIEPHFVDSAPVIEDDD


 


RKLPKLDEKTADSIRKGLTPKWNDLDVNQHVNNVKYIGWILESTPPEVLETQELCSLTL


 


EYRRECGRESVLESLTAVDPSGKGSGSQFQHLLRLEDGGEIVKGRTEWRPKTAGINGPIA


 


SGETSPGDSS


 


Chs FATB3 (SEQ ID NO: 19):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


RECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAIST


 


GKTSNGNSIS


 


ChsFatb3b (SEQ ID NO: 22):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHIEVNRYPTWGDTIEVNTWVSESGKTGMGR


 


DWLISDFHTGDILIRATSVCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKLH


 


KLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYRR


 


ECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAISTG


 


KTSNGNSIS


 


ChsFatB3c (SEQ ID NO: 106):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


QECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGTDIAKGRTKWRPKNAGKTSNGNS


 


IS


ChsFATB3d (SEQ ID NO: 107):


MVAAEASSALFSVRTPGTSPKPGKFGNWPSSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDASSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRSDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


RECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAIST


 


GKTSNGNSIS


 


ChsFATB3e (SEQ ID NO: 108):


MVAAEASSALFSVRTPGTSPKPGKFGNWPSSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDASSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRSDMLMDPFGVDRVVQDGVVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


RECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAIST


 


GKTSNGNSIS


 


ChsFATB3f (SEQ ID NO: 109):


MVAAEASSALFSVRTPGTSPKPGKFGNWPSSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


RECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAIST


 


GKTSNGNSIS


 


ChsFATB3g (SEQ ID NO: 110):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHIEVNRYPTWGDTIEVNTWVSESGKTGMGR


 


DWLISDFHTGDILIRATSVCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKLH


 


KLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYRQ


 


ECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGTDIAKGRTKWRPKNAGKTSNGNSIS


 


ChsFATB3h (SEQ ID NO: 111):


MVAAEASSALFSVRTPGTSPKPGKFGNWPSSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDASSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRSDMLMDPFGVDRVVQDGVVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHIEVNRYPTWGDTIEVNTWVSESGKTGMGR


 


DWLISDFHTGDILIRATSVCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKLH


 


KLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYRQ


 


ECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGTDIAKGRTKWRPKNAGKTSNGNSIS


 


ChsFATB3i (SEQ ID NO: 112):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHVEVNRYPTWGDTIEVNTWVSESGKTGMG


 


RDWLISDCHTGEILIRATSMCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKL


 


HKLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYR


 


RECGGDSVLESVTAMDPSKEGDRSLYQHLLRLEDGADIAKGRTKWRPKNAGTNGAIST


 


GKTSNGNSIS


 


ChsFATB3j (SEQ ID NO: 113):


MVAAEASSALFSVRTPGTSPKPGKFGNWPTSLSVPFKSKSNHNGGFQVKANASA


 


RPKANGSAVSLKSGSLDTQEDTSSSSSPPRTFINQLPDWSMLLSAITTVFVAAEKQWTML


 


DRKSKRPDMLMDPFGVDRVVQDGAVFRQSFSIRSYEIGADRTASIETLMNIFQETSLNHC


 


KSIGLLNDGFGRTPEMCKRDLIWVVTKMHIEVNRYPTWGDTIEVNTWVSESGKTGMGR


 


DWLISDFHTGDILIRATSVCAMMNQKTRRFSKFPYEVRQELAPHFVDSAPVIEDYQKLH


 


KLDVKTGDSICNGLTPRWNDLDVNQHVNNVKYIGWILESVPTEVFETQELCGLTLEYRQ


 


ECGRDSVLESVTAMDPSKEGDRSLYQHLLRLEDGTDIAKGRTKWRPKNAGKTSNGNSIS









EXAMPLE 2
In the example below, we detail the effect of expressing plant oilseed transcriptome-derived, heterologous thioesterases in the UTEX1435 (web.biosci.utexas.edu/utex/) strain, Strain A.
As in Example 1, RNA was extracted from dried plant seeds and submitted for paired-end sequencing using the Illumina Hiseq 2000 platform. RNA sequence reads were assembled into corresponding seed transcriptomes using Trinity or Oases packages and putative thioesterase-containing cDNA contigs were identified by mining transcriptomes for sequences with homology to known thioesterases. These in silico identified putative thioesterase cDNAs were verified by direct reverse transcription PCR analysis using seed RNA and primer pairs targeting full-length thioesterase cDNAs. The resulting amplified products were cloned and sequenced de novo to confirm authenticity of identified thioesterase genes and to identify sequence variants arising from expression of different gene alleles or diversity of sequences within a population of seeds. The resulting amino acid sequences were subjected to phylogenetic analysis using published full-length (Mayer and Shanklin, 2007) and truncated (THYME database) FatB sequences. The thioesterases that clustered with acyl-ACP FatB thioesterases, which are involved in biosynthesis of C8-C16 fatty acids, were pursued.
Construction of Transforming Vectors Expressing Acyl-ACP FatB Thioesterases
27 putative acyl-ACP FatB thioesterases from the species Cinnamomum camphora, Cuphea hyssopifolia, Cuphea PSR23, Cuphea wrightii, Cuphea heterophylla, and Cuphea viscosissima were synthesized in a codon-optimized form to reflect Prototheca moriformis (UTEX 1435) codon usage. Of the 27 genes synthesized, 24 were identified by our transcriptome sequencing efforts and the 3 genes from Cuphea viscosissima, were from published sequences in GenBank.
Transgenic strains were generated via transformation of the base strain Strain A (Prototheca moriformis, derived from UTEX 1435 by classical mutation and screening for high oil production) with a construct encoding 1 of the 27 FatB thioesterases. The construct pSZ2760 encoding Cinnamomum camphora (Cc) FATB1b is shown as an example, but identical methods were used to generate each of the remaining 26 constructs encoding the different respective thioesterases. Construct pSZ2760 can be written as 6S::CrTUB2: ScSUC2: CvNR::PmAMT3: CcFATB1b:CvNR::6S. The sequence of the transforming DNA is provided in Table 5 (pSZ2760). The relevant restriction sites in the construct from 5′-3′, BspQ1, KpnI, AscI, MfeI, EcoRI, SpeI, XhoI, SacI, BspQ1, respectively, are indicated in lowercase, bold, and underlined. BspQ1 sites delimit the 5′ and 3′ ends of the transforming DNA. Bold, lowercase sequences at the 5′ and 3′ end of the construct represent genomic DNA from UTEX 1435 that target integration to the 6S locus via homologous recombination. Proceeding in the 5′ to 3′ direction, the selection cassette has the C. reinhardtii f3-tubulin promoter driving expression of the S. cerevisiae gene SUC2 (conferring the ability to grow on sucrose) and the Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. The promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for ScSUC2 are indicated by bold, uppercase italics, while the coding region is indicated with lowercase italics. The 3′ UTR is indicated by lowercase underlined text. The spacer region between the two cassettes is indicated by upper case text. The second cassette containing the codon optimized CcFATB1b gene (Table 5; pSZ2760) from Cinnamomum camphora is driven by the Prototheca moriformis endogenous AMT3 promoter, and has the Chlorella vulgaris Nitrate Reductase (NR) gene 3′ UTR. In this cassette, the AMT3 promoter is indicated by lowercase, boxed text. The initiator ATG and terminator TGA for the CcFATB1b gene are indicated in bold, uppercase italics, while the coding region is indicated by lowercase italics and the spacer region is indicated by upper case text. The 3′ UTR is indicated by lowercase underlined text. The final construct was sequenced to ensure correct reading frame and targeting sequences.







TABLE 5





pSZ2760 Transforming construct (SEQ ID NO: 114)















gctcttcgccgccgccactcctgctcgagcgcgcccgcgcgtgcgccgccagcgccttggccttttcgccgcgctcgtgcgcgtcgct


 


gatgtccatcaccaggtccatgaggtctgccttgcgccggctgagccactgcttcgtccgggcggccaagaggagcatgagggag


 


gactcctggtccagggtcctgacgtggtcgcggctctgggagcgggccagcatcatctggctctgccgcaccgaggccgcctccaa


 


ctggtcctccagcagccgcagtcgccgccgaccctggcagaggaagacaggtgaggggggtatgaattgtacagaacaaccacg


 


agccttgtctaggcagaatccctaccagtcatggctttacctggatgacggcctgcgaacagctgtccagcgaccctcgctgccgcc


 


gcttctcccgcacgcttctttccagcaccgtgatggcgcgagccagcgccgcacgctggcgctgcgcttcgccgatctgaggacagt


 


cggggaactctgatcagtctaaacccccttgcgcgttagtgttgccatcctttgcagaccggtgagagccgacttgttgtgcgccacc


 


ccccacaccacctcctcccagaccaattctgtcacctttttggcgaaggcatcggcctcggcctgcagagaggacagcagtgccca


 








 








 








 








 


ctgcaggccttcctgttcctgctggccggcttcgccgccaagatcagcgcctccatgacgaacgagacgtccgaccgccccctggtgca


 


cttcacccccaacaagggctggatgaacgaccccaacggcctgtggtacgacgagaaggacgccaagtggcacctgtacttccagt


 


acaacccgaacgacaccgtctgggggacgcccttgttctggggccacgccacgtccgacgacctgaccaactgggaggaccagccc


 


atcgccatcgccccgaagcgcaacgactccggcgccttctccggctccatggtggtggactacaacaacacctccggcttcttcaacga


 


caccatcgacccgcgccagcgctgcgtggccatctggacctacaacaccccggagtccgaggagcagtacatctcctacagcctgga


 


cggcggctacaccttcaccgagtaccagaagaaccccgtgctggccgccaactccacccagttccgcgacccgaaggtcttctggtac


 


gagccctcccagaagtggatcatgaccgcggccaagtcccaggactacaagatcgagatctactcctccgacgacctgaagtcctgg


 


aagctggagtccgcgttcgccaacgagggcttcctcggctaccagtacgagtgccccggcctgatcgaggtccccaccgagcaggac


 


cccagcaagtcctactgggtgatgttcatctccatcaaccccggcgccccggccggcggctccttcaaccagtacttcgtcggcagcttc


 


aacggcacccacttcgaggccttcgacaaccagtcccgcgtggtggacttcggcaaggactactacgccctgcagaccttcttcaaca


 


ccgacccgacctacgggagcgccctgggcatcgcgtgggcctccaactgggagtactccgccttcgtgcccaccaacccctggcgctc


 


ctccatgtccctcgtgcgcaagttctccctcaacaccgagtaccaggccaacccggagacggagctgatcaacctgaaggccgagcc


 


gatcctgaacatcagcaacgccggcccctggagccggttcgccaccaacaccacgttgacgaaggccaacagctacaacgtcgacc


 


tgtccaacagcaccggcaccctggagttcgagctggtgtacgccgtcaacaccacccagacgatctccaagtccgtgttcgcggacct


 


ctccctctggttcaagggcctggaggaccccgaggagtacctccgcatgggcttcgaggtgtccgcgtcctcctcttcctggaccgcgg


 


gaacagcaaggtgaagttcgtgaaggagaacccctacttcaccaaccgcatgagcgtgaacaaccagcccttcaagagcgagaac


 


gacctgtcctactacaaggtgtacggcttgctggaccagaacatcctggagctgtacttcaacgacggcgacgtcgtgtccaccaacac


 


ctacttcatgaccaccgggaacgccctgggctccgtgaacatgacgacgggggtggacaacctgttctacatcgacaagttccaggtg


 








 


acttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgtttgatcttgtgtgtacgcgcttttgcgagttgctagctgc


 


ttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcatcccaaccgcaacttatctacgctgtcctgctatccct


 


cagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgcctgtattctcctggtactgcaacctgtaaaccagcac


 








 








 








 








 








 








 








 








 








 








 








 








 


catgaaggccgtgatgctggcccgcgacggccgcggcctgaagccccgctcctccgacctgcagctgcgcgccggcaacgcccaga


 


cctccctgaagatgatcaacggcaccaagttctcctacaccgagtccctgaagaagctgcccgactggtccatgctgttcgccgtgatc


 


accaccatcttctccgccgccgagaagcagtggaccaacctggagtggaagcccaagcccaaccccccccagctgctggacgacca


 


cttcggcccccacggcctggtgttccgccgcaccttcgccatccgctcctacgaggtgggccccgaccgctccacctccatcgtggccgt


 


gatgaaccacctgcaggaggccgccctgaaccacgccaagtccgtgggcatcctgggcgacggcttcggcaccaccctggagatgt


 


ccaagcgcgacctgatctgggtggtgaagcgcacccacgtggccgtggagcgctaccccgcctggggcgacaccgtggaggtgga


 


gtgctgggtgggcgcctccggcaacaacggccgccgccacgacttcctggtgcgcgactgcaagaccggcgagatcctgacccgct


 


gcacctccctgtccgtgatgatgaacacccgcacccgccgcctgtccaagatccccgaggaggtgcgcggcgagatcggccccgcct


 


tcatcgacaacgtggccgtgaaggacgaggagatcaagaagccccagaagctgaacgactccaccgccgactacatccagggcg


 


gcctgaccccccgctggaacgacctggacatcaaccagcacgtgaacaacatcaagtacgtggactggatcctggagaccgtgccc


 


gactccatcttcgagtcccaccacatctcctccttcaccatcgagtaccgccgcgagtgcacccgcgactccgtgctgcagtccctgacc


 


accgtgtccggcggctcctccgaggccggcctggtgtgcgagcacctgctgcagctggagggcggctccgaggtgctgcgcgccaag


 


accgagtggcgccccaagctgtccttccgcggcatctccgtgatccccgccgagtcctccgtgatggactacaaggaccacgacggcg


 


actacaaggaccacgacatcgactacaaggacgacgacgacaagTGActcgaggcagcagcagctcggatagtatcgacacact


 


ctggacgctggtcgtgtgatggactgttgccgccacacttgctgccttgacctgtgaatatccctgccgcttttatcaaacagcctcagtgtgttt


 


gatcttgtgtgtacgcgcttttgcgagttgctagctgcttgtgctatttgcgaataccacccccagcatccccttccctcgtttcatatcgcttgcat


 


cccaaccgcaacttatctacgctgtcctgctatccctcagcgctgctcctgctcctgctcactgcccctcgcacagccttggtttgggctccgc


 


ctgtattctcctggtactgcaacctgtaaaccagcactgcaatgctgatgcacgggaagtagtgggatgggaacacaaatggaAAGCT


 


GTATAGGGATAACAGGGTAATgagctcttgttttccagaaggagttgctccttgagcctttcattctcagcctcgata


 


acctccaaagccgctctaattgtggagggggttcgaatttaaaagcttggaatgttggttcgtgcgtctggaacaagcccagacttgt


 


tgctcactgggaaaaggaccatcagctccaaaaaacttgccgctcaaaccgcgtacctctgctttcgcgcaatctgccctgttgaaa


 


tcgccaccacattcatattgtgacgcttgagcagtctgtaattgcctcagaatgtggaatcatctgccccctgtgcgagcccatgcca


 


ggcatgtcgcgggcgaggacacccgccactcgtacagcagaccattatgctacctcacaatagttcataacagtgaccatatttctc


 


gaagctccccaacgagcacctccatgctctgagtggccaccccccggccctggtgcttgcggagggcaggtcaaccggcatgggg


 


ctaccgaaatccccgaccggatcccaccacccccgcgatgggaagaatctctccccgggatgtgggcccaccaccagcacaacct


 


gctggcccaggcgagcgtcaaaccataccacacaaatatccttggcatcggccctgaattccttctgccgctctgctacccggtgctt


 


ctgtccgaagcaggggttgctagggatcgctccgagtccgcaaacccttgtcgcgtggcggggcttgttcgagcttgaagagc









Constructs encoding the identified heterologous FatB genes, such as CcFATB1b from pSZ2760 in Table 6, were transformed into Strain A, and selected for the ability to grow on sucrose. Transformations, cell culture, lipid production and fatty acid analysis were all carried out as previously described. After cultivating on sucrose under low nitrogen conditions to accumulate oil, fatty acid profiles were determined by FAME-GC. The top performer from each transformation, as judged by the ability to produce the highest level of midchain fatty acids, is shown in Table 4.







TABLE 6







Alteration of Fatty Acid Profiles in S3150 upon Expression of Heterologous FatB


Thioesterases










SZ
FA profile of top performer from each transformation (%; primary lipid in Strain A background)



















Species
Gene Name
Plasmid
Strain
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1
C18:2
C18:3α






















Cinnamomum
CcFATB1b
pSZ2760
A; T526;
0
0
1
15
26
2
46
9
1


camphora


D1670-13


Cinnamomum
CcFATB4
pSZ2756
A; T525;
0
1
33
4
7
2
41
10
1


camphora


D1666-31


Cinnamomum
CcFATB3
pSZ2755
A; T525;
0
0
0
3
44
3
41
8
0


camphora


D1665-4


Cuphea hyssopifolia
ChsFATB1
pSZ2778
A; T535;
0
0
0
2
22
4
63
8
1





D1689-30


Cuphea hyssopifolia
ChsFATB2
pSZ2796
A; T537;
0
0
0
6
53
3
32
6
0





D1700-46


Cuphea hyssopifolia
ChsFATB2b
pSZ2792
A; T537;
0
0
0
5
26
2
56
9
1





D1696-9


Cuphea hyssopifolia
ChsFATB3
pSZ2797
A; T537;
0
0
8
34
27
2
24
5
1





D1701-48


Cuphea hyssopifolia
ChsFATB3b
pSZ2795
A; T537;
0
0
7
29
27
1
28
6
1





D1699-1


Cuphea PSR23
CuPSR23FATB3
pSZ2793
A; T537;
0
1
0
2
24
3
61
8
1





D1697-13


Cuphea wrightii
CwFATB3
pSZ2751
A; T525;
0
2
17
9
19
2
41
8
1





D1661-22


Cuphea wrightii
CwFATB4a
pSZ2752
A; T525;
0
0
0
4
48
3
36
7
1





D1662-30


Cuphea wrightii
CwFATB4b
pSZ2753
A; T525;
0
0
0
5
52
3
32
6
1





D1663-29


Cuphea wrightii
CwFATB5
pSZ2754
A; T525;
0
0
0
3
27
3
57
7
1





D1664-39


Cuphea heterophylla
ChtFATB1a
pSZ2757
A; T525;
0
0
5
18
27
2
39
7
1





D1667-19


Cuphea heterophylla
ChtFATB1b
pSZ2773
A; T535;
0
0
2
7
27
3
53
8
1





D1685-29


Cuphea heterophylla
ChtFATB2b
pSZ2780
A; T535;
0
0
0
2
25
3
61
8
1





D1691-8


Cuphea heterophylla
ChtFATB2a
pSZ2774
A; T537;
0
0
0
2
27
3
59
6
0





D1702-24


Cuphea heterophylla
ChtFATB2c
pSZ2758
A; T525;
0
0
3
2
23
3
58
7
1





D1668-22


Cuphea heterophylla
ChtFATB2d
pSZ2759
A; T526;
0
0
4
4
23
3
54
9
1





D1669-19


Cuphea heterophylla
ChtFATB2e
pSZ2775
A; T535;
0
1
2
3
24
3
57
8
1





D1686-23


Cuphea heterophylla
ChtFATB2f
pSZ2777
A; T535;
0
0
0
2
28
3
57
8
1





D1688-33


Cuphea heterophylla
ChtFATB2g
pSZ2794
A; T537;
0
0
0
2
22
3
62
9
1





D1698-19


Cuphea heterophylla
ChtFATB3a
pSZ2776
A; T535;
0
0
0
5
47
4
37
7
1





D1687-23


Cuphea heterophylla
ChtFATB3b
pSZ2779
A; T535;
0
0
0
6
49
5
32
7
0





D1690-31


Cuphea viscosissima
CvisFATB1
pSZ2810
A; T540;
0
1
0
2
24
3
60
8
0





D1711-30


Cuphea viscosissima
CvisFATB2
pSZ2817
A; T547;
0
0
0
4
51
2
36
6
0





D1718-1


Cuphea viscosissima
CvisFATB3
pSZ2791
A; T537;
0
0
0
8
28
2
52
8
1





D1695-1





A (parent
0
0
0
2
28
3
58
7
0





strain):









Many of the acyl-ACP FatB thioesterases were found to exhibit midchain activity when expressed in Prototheca moriformis. For example, expression of CcFATB1b causes an increase in myristate levels from 2% of total fatty acids in the parent, Strain A, to  ̃15% in the D1670-13 primary transformant. Other examples include CcFATB4, which exhibits an increase in laurate levels from 0% in Strain A to  ̃33%, and ChsFATB3, which exhibits an increase in myristate levels to  ̃34%. Although some of the acyl-ACP thioesterases did not exhibit dramatic effects on midchain levels in the current incarnation, efforts will likely develop to optimize some of these constructs.
Sequences of the Heterologous Acyl-ACP Thioesterases Identified and Transformed into P. moriformis (UTEX 1435)
A complete listing of relevant sequences for the transforming constructs, such as the deduced amino acid sequence of the encoded acyl-ACP thioesterase, the native CDS coding sequence, the Prototheca moriformis codon-optimized coding sequence, and the nature of the sequence variants examined, is provided as SEQ ID NOS: 1-78.

## Claims

1. A recombinant nucleic acid having at least 97% sequence identity to any of SEQ ID NOS: 5 or 6, or any equivalent sequences by virtue of the degeneracy of the genetic code, wherein the recombinant nucleic acid encodes a protein having acyl-ACP thioesterase activity.

2. A recombinant nucleic acid encoding a protein having at least 90% sequence identity to SEQ ID NO: 4 and acyl-ACP thioesterase activity, wherein the recombinant nucleic acid also comprises a heterologous nucleic acid.

3. A method of producing a recombinant cell, the method comprising transforming the cell with a nucleic acid according to any of claim 1 or 2.

4. A host cell comprising a recombinant nucleic acid encoding a protein having at least 90% sequence identity to SEQ ID NO:4 and acyl-ACP thioesterase activity, wherein the recombinant nucleic acid comprises an exogenous nucleic acid, and wherein the host cell has an altered fatty acid profile.

5. The host cell of claim 4, wherein the host cell is selected from a plant cell, a microbial cell, and a microalgal cell.

6. A method for producing an oil or oil-derived product, the method comprising cultivating a host cell of claim 4 and extracting oil produced thereby, optionally wherein the cultivation is heterotrophic growth on sugar.

7. The method of claim 6, further comprising producing a fatty acid, fuel, chemical, or other oil-derived product from the oil.

8. A vector comprising the recombinant nucleic acid of claim 1 or 2.